Use of AgomiR and AntagomiR technologies to alter satellite cell proliferation in vitro, miRNA expression, and muscle fiber hypertrophy in intrauterine growth-restricted lambs.

Introduction: microRNAs (miRNAs) are small non-coding RNAs that work at the posttranscriptional level to repress gene expression. Several miRNAs are preferentially expressed in skeletal muscle and participate in myogenesis. This research was conducted to alter endogenous miRNA expression in skeletal muscle to promote muscle hypertrophy. Methods: Two experiments were conducted using mimic/agomiR or antagomir technologies to alter miRNA expression and examine changes in myoblast proliferation in vitro (experiment 1) and muscle hypertrophy in vivo (experiment 2). In vitro experiments found that antagomiR-22-3p and mimic-127 increased myoblast proliferation compared to other miRNA treatments or controls. These miRNA treatments, antagomiR-22-3p (ANT22) and agomiR-127 (AGO127), were then used for intramuscular injections in longissimus muscle. Results and discussion: The use of antagomiR or mimic/agomiR treatments down-regulated or up-regulated, respectively, miRNA expression for that miRNA of interest. Expression of predicted target KIF3B mRNA for miR-127 was up-regulated and ACVR2a mRNA was up-regulated for miR-22-3p. ANT22 injection also up-regulated the major regulator of protein synthesis (mTOR). Proteomic analyses identified 11 proteins for AGO127 and 9 proteins for ANT22 that were differentially expressed. Muscle fiber type and cross-sectional area were altered for ANT22 treatments to transition fibers to a more oxidative state. The use of agomiR and antagomir technologies allows us to alter miRNA expression in vitro and in vivo to enhance myoblast proliferation and alter muscle fiber hypertrophy in IUGR lambs during early postnatal growth.

Mycotoxin ingestion during late gestation alters placentome structure, cotyledon transcriptome, and fetal development in pregnant sheep.

Ergot alkaloids, a class of mycotoxins, induce vasoconstriction when consumed by animals and humans. Pregnant ewes (n = 16; 81.2 kg ± 7.7) were assigned fed endophyte-infected tall fescue seed (E+; 4.14 µg ergovaline + ergovalinine/g seed) or a control diet (CON; 0 µg ergovaline + ergovalinine) for increasing duration during late gestation (from gd86 to gd110 or gd133) to examine changes in placentome morphology and mRNA transcriptome, and fetal development. Exposure to E+ fescue reduced serum prolactin concentrations at gd110 and gd133 demonstrating treatment efficacy. For control ewes, cotyledon and total placentome weights decreased with advancing gestation due to remodeling of placental tissues; however, cotyledon and placentome weight did not change with advancing gestation in E+ fed ewes. Fetal brain sparing was evident in E+ exposed fetuses at gd110 and gd133 compared to CON, which demonstrates asymmetrical growth and intrauterine growth restriction. Mycotoxin exposure (E+) resulted in differential expression of 22 genes in the cotyledon tissue at gd110 but only one gene at gd133 compared to CON. These results suggest that the response to mycotoxin exposure in the pregnant sheep model has an immediate impact on placental remodeling and fetal development that persists throughout the duration of the exposure period.

Residual effects of abomasal 5-hydroxytryptophan administration on serotonin metabolism in cattle.

Studies of serotonin in animal husbandry has received growing interest. However, there is limited information about serotonin manipulation using 5-HTP administered postruminally and its residual effects in cattle. The objective of this study was to evaluate the effectiveness of 5-HTP infused into the abomasum for enhancing circulating serotonin in cattle. Four Holstein steers (487 ± 7.6 kg) fitted with ruminal cannulas were used in a 4 × 4 Latin Square design experiment. The treatments were intra-abomasal infusion of 5-HTP at 0, 0.25, 0.5, and 1 mg/kg BW. Blood was collected from the jugular vein of each steer at -60, -30, 0, 30, 60, 120, 240, and 480 min from 5-HTP infusion for basal and short term evaluation and, at 1, 2, 4, and 7 d after 5-HTP infusion for long term evaluation. Dry matter intake was not affected (P > 0.05) by intra-abomasal infusions. The half-life of 5-HTP was dose-independent (128 min). The serum 5-HTP, serotonin, and 5-hydroxyindoleacetic acid area under the curve increased (P < 0.05) linearly with an increased dose of 5-HTP. Serum 5-HTP reached peak concentration in approximately 30 min after dosing while serum and plasma serotonin peaked after 240 min postinfusion. Serotonin was greater than control for all 5-HTP doses 1 d and 2 d after infusion in serum and plasma, respectively. Intra-abomasal infusion of 5-HTP at doses up to 1 mg/ kg BW increases circulating serotonin for up 2 days.

Robust Fermi-Surface Morphology of CeRhIn_{5} across the Putative Field-Induced Quantum Critical Point.

We report a comprehensive de Haas-van Alphen (dHvA) study of the heavy-fermion material CeRhIn_{5} in magnetic fields up to 70 T. Several dHvA frequencies gradually emerge at high fields as a result of magnetic breakdown. Among them is the thermodynamically important ß_{1} branch, which has not been observed so far. Comparison of our angle-dependent dHvA spectra with those of the non-4f compound LaRhIn_{5} and with band-structure calculations evidences that the Ce 4f electrons in CeRhIn_{5} remain localized over the whole field range. This rules out any significant Fermi-surface reconstruction, either at the suggested nematic phase transition at B^{*}≈30 T or at the putative quantum critical point at B_{c}≃50 T. Our results rather demonstrate the robustness of the Fermi surface and the localized nature of the 4f electrons inside and outside of the antiferromagnetic phase.

Pattern of postruminal administration of l-tryptophan affects blood serotonin in cattle.

Serotonin (5-HT) has many important functions in both central and peripheral nervous systems. Although it has been demonstrated that manipulation of serotonin metabolism is possible in many species, there is limited information about l-tryptophan (TRP), a serotonin precursor, in cattle, and these provide conflicting results. Furthermore, there is no study evaluating how different patterns of intra-abomasal infusion of TRP impact circulating 5-HT. The objective of this study was to evaluate if intra-abomasal infusion patterns of TRP can affect circulating 5-HT and other metabolites from TRP metabolism in the plasma and serum and circulating glucose and insulin in cattle. Eight ruminally cannulated Holstein steers were used in a replicated 4 × 4 Latin square design. Each received intra-abomasal water infusion (control) or intra-abomasal TRP infusion (50 mg/kg BW) in 3 different patterns: a pulse infusion once a day (pulse once), pulse infusion twice a day (pulse twice), or continuous infusion (continuous). For continuous treatment, the TRP dose was diluted in tap water and infused by a peristaltic pump (300 mL/h). To equalize conditions, the other treatments had a water infusion (300 mL/h). The steers were fed every 2 h, and blood was collected from a jugular vein catheter every 4 h for 24 h after the initial infusion. Urine produced during the 24 h period was collected. Serum and plasma TRP, 5-HT and kynurenine, plasma glucose, and serum insulin concentrations were analyzed. Urine was analyzed for concentrations of 5-hydroxyindoleacetic acid. Both serum TRP and kynurenine were increased (P < 0.05) by all TRP infusion treatments, but concentrations in pulse dose treatments were greater than those in continuous infusion. Serum 5-HT increased (P < 0.05) with both pulse TRP infusion treatments; however, the continuous TRP infusion did not increase the serum 5-HT. Plasma 5-HT, glucose, and insulin had a tendency to increase with TRP pulse infusions. The urinary 5-hydroxyindoleacetic acid excretion was highest for pulse dose treatments. An acute supply of TRP in 1 or 2 daily doses increases serum 5-HT and increases circulating glucose and insulin in cattle. The TRP and kynurenine concentrations are similar in plasma and serum. However, the serum 5-HT concentration is more responsive to TRP administration than plasma.

5-Hydroxytryptophan strongly stimulates serotonin synthesis in Holstein steers.

Although serotonin has been extensively studied in many species, there is a lack of information in ruminants, and no research has been evaluated if its precursor, 5-hydroxytryptophan (5-HTP), administered into the abomasum may be used as a means to manipulate serotonin metabolism. Thus, the objective of this study was to evaluate if intra-abomasal infusion of 5-HTP increases circulating serotonin in the steer. Eight Holstein steers (471 ± 8.9 kg) were used in a replicated 4 × 4 Latin Square design experiment. The treatments were intra-abomasal infusion of 5-HTP at 0.5, 1, 2.5, and 5 mg/kg BW. Blood was collected at 0, 2, 4, 6, 8, and 24 h after infusion. The serum concentration of 5-HTP increased quadratically (P = 0.005) with a peak at 2 h after administration. The 5-HTP administration increased (P < 0.05) serum serotonin in comparison with baseline with no difference (P > 0.05) between the doses of 5-HTP. When 5-HTP was dosed at 2.5 mg/kg BW or higher, intake decreased, and there was an altered manure consistency. The serum 5-hydroxyindole acetic acid concentrations followed the same pattern as 5-HTP. Plasma glucose content was not affected (P > 0.05) by 5-HTP dosing. However, free fatty acids concentration in the plasma was lower (P > 0.05) compared with baseline for the infusion levels of 0.5 and 1 mg/kg BW. Intra-abomasal infusion of 5-HTP efficiently increases serum serotonin cattle.

Tall fescue ergot alkaloids are vasoactive in equine vasculature.

Mares grazing endophyte-infected () tall fescue () typically exhibit reproductive dysfunction rather than problems associated with peripheral vasoconstriction as a primary sign of the fescue toxicosis syndrome. Research using Doppler ultrasonography demonstrated that consumption of endophyte-infected tall fescue seed causes measurable vasoconstriction in the medial palmar artery. The objective of this study was to evaluate contractile responses of medial palmar artery and vein to increasing concentrations of various tall fescue alkaloids. Medial palmar arteries and veins were collected immediately following euthanasia from 23 horses of mixed breed, age, and gender from both forelimbs, and uterine arteries were collected from females ( = 12). Vessels were separated, cleaned of excess connective and adipose tissue, divided into 2- to 3-mm cross-sections, and suspended in a multimyograph chamber with continuously oxygenated Krebs-Henseleit buffer (95% O/5% CO; pH 7.4; 37°C). Following a 90-min equilibration and recovery from reference compound exposure, increasing concentrations of norepinephrine, 5-hydroxytryptamine, ergotamine, and ergonovine for the palmar artery and vein and uterine artery and ergovaline, ergocryptine, ergocristine, ergocornine, and lysergic acid for the palmar artery and vein were added to assess vasoactivity. Data were normalized as a percentage of contractile response induced by the reference compound addition and analyzed as a completely randomized design. Both norepinephrine and serotonin were vasoactive in all 3 types of blood vessels. Neither ergotamine nor ergonovine were vasoactive in the uterine artery. All alkaloids tested with the palmar artery and vein produced a contractile response, except that neither the palmar artery nor the palmar vein responded to lysergic acid ( > 0.05). Ergovaline was the most vasoactive ergot alkaloid in both the palmar artery and the palmar vein ( < 0.05) followed by ergonovine, whereas out of the 4 remaining ergopeptine alkaloids tested, ergocristine induced the lowest contractile response. Although horses do not outwardly appear to be affected by peripheral vasoconstriction as observed in cattle, these data indicate that tall fescue alkaloids are vasoactive and suggest that potential exists for peripheral vascular effects of tall fescue alkaloids in horses. This does not appear to be the case for the uterine artery, and future research should be directed at understanding how ergot alkaloids cause equine reproductive dysfunction.

Interaction between a tannin-containing legume and endophyte-infected tall fescue seed on lambs' feeding behavior and physiology.

It was hypothesized that a tannin-rich legume such as sainfoin attenuates the negative postingestive effects of ergot alkaloids in tall fescue. Thirty-two 4-mo-old lambs were individually penned and randomly assigned to a 2 × 2 factorial arrangement with 2 legume species, sainfoin (SAN; 2.9% condensed tannins) or cicer milkvetch (CIC; without tannins) and a mixed ration containing tall fescue seed (50:30:20 seed:beet pulp:alfalfa) with 2 levels of endophyte infection (endophyte-infected tall fescue seed [E+; 3,150 ug/L ergovaline] or endophyte-free tall fescue seed [E-]). For a 10-d baseline period, half of the lambs were fed SAN and half were fed CIC and all lambs had ad libitum amounts of E-. In an ensuing 10-d experimental period, the protocol was the same except half of the lambs fed SAN or CIC received E+ instead of E-. Subsequently, all lambs could choose between their respective legume and seed-containing ration and between E+ and E-. Finally, an in vitro radial diffusion assay was conducted to determine whether tannins isolated from SAN would bind to alkaloids isolated from E+. All groups consumed similar amounts of E- during baseline period ( > 0.10), but lambs ate more E- than E+ during the experimental period ( < 0.05) and lambs offered SAN ate more E+ than lambs offered CIC ( < 0.05). Groups fed E- during the baseline and experimental periods had similar rectal temperatures ( > 0.10), but lambs fed E+ had lower rectal temperatures per gram of feed ingested when supplemented with SAN than with CIC ( < 0.05). Lambs fed E+ had greater concentrations of hemoglobin and more red blood cells than lambs fed E- ( < 0.05), but plasmatic concentrations of cortisol and prolactin did not differ among treatments ( > 0.10). All lambs preferred their treatment ration over their treatment legume, but lambs in the SAN and E+ treatment ate more legume + ration than lambs in the CIC and E+ (CIC-E+; < 0.05) treatment. All lambs preferred E- over E+, but lambs in the CIC-E+ treatment ate the least amount of E+ ( < 0.05). Binding of isolated SAN tannins to protein was reduced by the E+ isolate ( < 0.05), suggesting a tannin-alkaloid complexation but only from tannins extracted from SAN fed early in the experimental period. In summary, SAN supplementation increased intake of and preference for E+ and reduced rectal temperatures relative to CIC supplementation. Our results suggest that these effects were mediated by the condensed tannins in SAN through alkaloid inactivation, an interaction that declined with plant maturity.

BILL E. KUNKLE INTERDISCIPLINARY BEEF SYMPOSIUM: Physiologic effects of ergot alkaloids: What happens when excretion does not equal consumption?

Increased persistence of tall fescue () infested with an endophytic fungus, (formerly ), in forage-based agriculture has led to increased effort in understanding the negative effects caused by consumption of ergot alkaloids by animals consuming this forage. Ergot alkaloids have been shown to have an extremely short plasma half-life, but this does not necessarily equate to total clearance. Studies that measured consumption and excretion of alkaloids have demonstrated that in the case of ergovaline, less is excreted than is consumed. The fate of ergot alkaloids that leave circulation but are not excreted is not well understood. Consequently, these "alkaloid balance studies" have led to speculation that ergovaline might bioaccumulate in the animal. Unfortunately, few data indisputably support this outcome. Progress has been slowed by the fact that the fungus produces a multitude of different ergot alkaloids that can bind to a variety of different receptors. Binding studies have shown that ergot alkaloids have unusually slow receptor dissociation rates that have been described as irreversible and contribute to a persistent signaling effect. In vitro analyses have revealed a potential for accumulation of ergot alkaloids through repetitive exposures to low concentrations creating a "depot" of alkaloids available to interact with receptors. The specific high binding affinity of ergot alkaloids combined with the potential turnover of alkaloids bound nonspecifically could extend residual effects of these compounds. Interestingly, cattle exposed to ergot alkaloids in vivo have a consistently lower vascular response to agonists that target receptors known to bind ergot alkaloids. If these same receptors are blocked with an antagonist, contractile response to ergopeptine alkaloids is also reduced significantly (>60% reduction). This observation that alkaloid exposure interrupts normal function of a receptor can persist 5 to 6 wk after animals have been removed from an ergot alkaloid source (and prolactin levels have long since returned to normal). Thus, clearance of ergot alkaloids from cattle grazing pasture with ergot alkaloid-producing endophytes may occur in a similar gradual manner. Studies that improve the understanding of how cattle process ergot alkaloids will help answer the question of whether ergot alkaloids bioaccumulate.

Combined approach in the treatment of chronic anal fissures.

This study was designed to evaluate the healing and complications rates in surgically and conservatively treated patients with chronic anal fissure. Conservative treatment consisted of nitrate or diltiazem ointment. In case of surgery, fissurectomy was performed. In total, 340 patients were included in the study. Among them, 162 patients had surgery and 178 patients had conservative treatment. The healing rate at surgically treated group of patients varied between 95 and 98% depending on previous treatment. Group treated with nitrate ointment and group treated with diltiazem ointment showed, respectively 62% and 52% healing rates. Difference between ointments was not statistically significant. Average healing time was between 105 and 123 days and complication rates were between 1.7 and 5.4%. The surgical treatment showed much higher healing rates and thus should recommended as primary treatment option for the chronic anal fissure, especially if there are chronic secondary lesions already present. In case of conservative treatment, either nitrate or diltiazem ointment could be used with similar efficacy.

Effect of intake on fasting heat production, respiratory quotient and plasma metabolites measured using the washed rumen technique.

The objective was to investigate the effect of intake before fasting on concentrations of metabolites and hormones, respiratory quotient (RQ) and fasting heat production (HP) using the washed rumen technique and to compare these values with those from the fed state. Six Holstein steers (360±22 kg) were maintained at 21°C and fed three different energy intakes within a replicated 3×3 Latin square design with 21-day periods. Steers were fed alfalfa cubes to provide 1.0, 1.5 and 2.0×NEm during 19 days of each experimental period. Steers were placed in individual metabolism stalls fitted with indirect calorimetry head-boxes on day 20 of each experimental period (FED steers) and fed their normal meal. On day 21 of each period the reticulorumen was emptied, washed and refilled with ruminal buffer (NaCl=96; NaHCO3=24; KHCO3=30; K2HPO4=2; CaCl2=1.5; MgCl2=1.5 mmol/kg of buffer) aerated with 75% N2 and 25% CO2 before introduction to the rumen (steers were not fed; WASHED steers). Each gas exchange was measured over 24 h. HP for 1.0, 1.5 and 2.0×NEm were 479, 597 and 714 kJ/daykg0.75 (s.e.m. =16), respectively. The plateau RQ was 0.756, 0.824 and 0.860 for the 1.0, 1.5 and 2.0×NEm intakes for the FED steers, respectively. After rumen washing, fasting HP was 331, 359 and 400 kJ/daykg0.75 (s.e.m.=13) for 1.0, 1.5, and 2.0×NEm intakes before fasting, respectively. The RQ for WASHED rumen steers was 0.717, 0.710 and 0.719, respectively. Cortisol and ß-hydroxybutyrate concentrations in WASHED rumen steers did not exceed threshold levels for severe energy deficit and stress as can be induced from prolonged fasting. This study demonstrates that a fasting state can be emulated using the washed rumen technique, minimizing the time required as opposed to traditional fasting methodologies, without causing a severe energy deficit and stress.

Providing systematic detailed information on medication upon hospital discharge as an important step towards improved transitional care.

WHAT IS KNOWN AND OBJECTIVE: Good communication between hospital and primary care physicians (PCPs) is important for the continuity of patient care in the transition phase following hospital treatment. Drug-related problems and medication errors may occur at that interface. Discharge letters often lack a structured medication report at the end and therefore may not provide the reasons for medication changes, resulting in low adherence rates. The objectives were to develop a structured medication report as part of the discharge letter, to evaluate the impact of the medication report in ischaemic stroke patients and to identify the most important issues in the transitional care process of stroke patients. METHODS: First, a structured medication report was developed. Thereafter, the impact of this new medication report on clinical practice was evaluated with an open, prospective, interventional two-phase study conducted at the Klinikum Fulda gAG (Germany), which included patients with ischaemic stroke and >2 drugs in the discharge medication. In the control group (CG), the neurologist included the current medication in the discharge letter. In the intervention group (IG), the clinical pharmacist added the detailed information to a medication report. To evaluate adherence to discharge medication, the PCP was interviewed 3 months after hospital discharge about the medication. Adherence was measured with respect to the entire medication regimen, antithrombotic and cholesterol-lowering drugs and discontinued medication. The most important issues in the transitional care for patients with ischaemic stroke were identified on the basis of the secondary stroke prevention and cardiovascular risk factors. RESULTS AND DISCUSSION: Overall, 312 patients were enrolled in the study with 156 patients in each group. By providing detailed information in the newly developed discharge letter, adherence increased significantly from 83·3% (CG) to 90·9% (IG; P = 0·01). Significant differences between the CG and IG were found with regard to adherence to both antithrombotic drugs [83·8% CG vs. 91·9% IG (P = 0·033)] and statin therapy [69·8% CG vs. 87·7% IG (P < 0·001)]. WHAT IS NEW AND CONCLUSION: The use of a structured medication report as part of the discharge letter leads to improved adherence to hospital discharge medication.

Bovine lateral saphenous veins exposed to ergopeptine alkaloids do not relax.

The ergot alkaloid ergovaline has demonstrated a persistent and sustained contractile response in several different vascular models. It was hypothesized that different alkaloids isolated from tall fescue (Lolium arundinaceum) will contribute to this contractile response differently. The objective was to compare contractile-response patterns of single additions of the ergoline alkaloids lysergic acid, lysergol, and ergonovine and the ergopeptine alkaloids ergotamine, ergocristine, ergocryptine, ergocornine, and ergovaline (provided as tall fescue seed extract). Lateral saphenous veins were collected from 6 Holstein steers (BW = 397 ± 28 kg) immediately after slaughter, sliced into cross-sections, and suspended in myograph chambers containing oxygenated Krebs-Henseleit buffer (95% O2/5% CO2; pH = 7.4; 37°C). Treatments were added at 0 min and buffer was replaced in 15-min intervals for a 120-min incubation. In addition to maximum tension and time to reach maximum tension, percent relaxation and rate of relaxation were determined following maximum tension for each treatment. All compounds tested produced significant contractile responses (P < 0.05). All ergoline alkaloids reached maximum response in less time (P < 0.05) than the remaining compounds and began to relax immediately after first buffer change. Lysergic acid had the greatest (P < 0.05) percent relaxation and ergonovine had the greatest (P < 0.05) rate of relaxation. The ergopeptine alkaloids ergovaline, ergotamine, ergocristine, ergocryptine, and ergocornine had slower developing contractile responses with a longer (P < 0.05) interval until maximum tension was achieved compared to the ergoline alkaloids. Maximal responses to all the ergopeptine alkaloids, however, all persisted for the 120-min duration with negligible relaxation occurring. The different classes of alkaloids differed greatly in the type of contractile response generated in the lateral saphenous vein. Persistence of contractile response is thought to be the primary contributing factor to the vasoconstriction observed in animals demonstrating signs of fescue toxicosis, where different ergot alkaloids can contribute differently.

Dietary exposure to ergot alkaloids decreases contractility of bovine mesenteric vasculature.

Ergot alkaloids are hypothesized to cause vasoconstriction in the midgut, and prior exposure may affect the vasoactivity of these compounds. The objectives of this study were to profile vasoactivity of ergot alkaloids in bovine mesenteric artery (MA) and vein (MV) and determine if previous exposure to endophyte-infected tall fescue seed affected vasoactivity of ergocryptine (ERP), ergotamine (ERT), ergocristine (ERS), ergocornine (ERO), ergonovine (ERN), lysergic acid (LSA), ergovaline-containing tall fescue seed extract (EXT), and 5-hydroxytryptamine (5HT; serotonin). Ruminally cannulated Angus steers (n = 12; BW = 547 ± 31 kg) were paired by weight and randomly assigned to 6 blocks. Steers were ruminally dosed daily with 1 kg of either endophyte-infected (E+; 4.45 mg ergovaline/kg DM) or endophyte-free (E-; 0 mg ergovaline/kg DM) tall fescue seed for 21 d before slaughter. Branches of MA and MV supporting the cranial portion of the ileum were collected after slaughter on d 22, placed in a modified Krebs-Henseleit buffer on ice, cleaned, sectioned, and mounted in a multimyograph chamber. Contractile response was normalized to a maximum KCl response. Inner diameter (P = 0.04) and outer diameter (P = 0.02) of MA were smaller for E+ steers than E- steers. Maximum contractile responses to 120 mM KCl were not different between seed treatments in MA (P = 0.33; E-: 2.67 ± 0.43 g; E+: 3.33 ± 0.43 g) or MV (P = 0.26; E-: 2.01 ± 0.18 g; E+: 1.81 ± 0.18 g). Steers receiving E+ had a smaller (P < 0.01) MA contractile response than E- steers to ERP, ERT, ERS, ERO, ERN, EXT, and 5HT. Steers receiving E+ had a smaller (P < 0.05) MV contractile response than E- steers to ERP, ERT, ERS, ERN, EXT, and 5HT. Lysergic acid failed to induce a contractile response in MA and MV. The contractile response in MA and MV of E- steers produced by 5HT was very large. The EXT was the most potent (P < 0.05) agonist in MV and MA of E+ steers. These data showed that ergot alkaloids were vasoactive in the bovine midgut, and steers exposed to E+ had diminished contractility to some ergot alkaloids in small intestinal vasculature. The findings of this study suggest that dietary exposure to ergot alkaloids has the potential to alter nutrient absorption from the midgut by decreasing blood flow to and from the midgut due to vasoconstriction.

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.

Tall fescue seed extraction and partial purification of ergot alkaloids.

Many substances in the tall fescue/endophyte association (Schedonorus arundinaceus/Epichloë coenophiala) have biological activity. Of these compounds only the ergot alkaloids are known to have significant mammalian toxicity and the predominant ergot alkaloids are ergovaline and ergovalinine. Because synthetically produced ergovaline is difficult to obtain, we developed a seed extraction and partial purification protocol for ergovaline/ergovalinine that provided a biologically active product. Tall fescue seed was ground and packed into several different sized columns for liquid extraction. Smaller particle size and increased extraction time increased efficiency of extraction. Our largest column was a 114 × 52 × 61 cm (W × L × D) stainless steel tub. Approximately 150 kg of seed could be extracted in this tub. The extraction was done with 80% ethanol. When the solvent front migrated to bottom of the column, flow was stopped and seed was allowed to steep for at least 48 h. Light was excluded from the solvent from the beginning of this step to the end of the purification process. Following elution, ethanol was removed from the eluate by evaporation at room temperature and the resulting syrup was freeze-dried. About 80% recovery of alkaloids was achieved with 18-fold increase in concentration of ergovaline. Initial purification of the dried product was accomplished by extracting with hexane/water (6:1, v/v). The aqueous fraction was extracted with chloroform, the aqueous layer discarded, after which the chloroform was removed with a resulting 20-fold increase of ergovaline. About 65% of the ergovaline was recovered from the chloroform residue for an overall recovery of 50%. The resultant partially purified ergovaline had biological activities in in vivo and in vitro bovine bioassays that approximate that of synthetic ergovaline.

Postgraze assessment of toxicosis symptoms for steers grazed on toxic endophyte-infected tall fescue pasture.

A 2-yr pen experiment was conducted using 12 different crossbred Angus steers each year to determine if short-term changes in prolactin concentrations, body temperature, and vasoconstriction reflect recovery from fescue toxicosis after steers that previously grazed toxic endophyte (Neotyphodium coenophialum)-infected Kentucky 31 tall fescue [Lolium arundinaceum (Schreb.) Darbysh] are placed on nontoxic feed. Groups of 6 steers from toxic endophyte-infected and endophyte-free tall fescue grazing treatments were blocked by BW for assignment to pens as a randomized complete block design with 2 replications. Two environments were implemented by initiating the experiment on 18 August in yr 1 and on 8 September in yr 2 for durations of 30 and 21 d, respectively. Rectal temperatures were recorded, jugular blood was collected for assaying serum prolactin, and cross sections of the caudal artery were ultrasonically imaged at selected time points to evaluate temporal changes in the response variables. Rectal temperatures in steers on the toxic endophyte pasture treatment declined (P < 0.05) linearly over time in yr 1 and 2 and were similar (P > 0.10) to those on endophyte-free treatment on d 30 in yr 1 and by d 15 in yr 2. Prolactin concentrations in steers on the toxic endophyte pasture treatment showed curvilinear increases (P < 0.05) over time and were similar (P > 0.10) to steers on the endophyte-free treatment by d 15 in yr 1 and by d 10 in yr 2. Luminal areas of the caudal artery in toxic endophyte steers were less (P < 0.05) than those in endophyte-free steers across all dates in both years. Results indicated that rectal temperatures in steers after they are removed from toxic fescue may decrease over time, but temporal changes in rectal temperatures could be affected more by prevailing ambient temperatures than by actual mitigation of fescue toxicosis. Prolactin concentrations in steers after they are removed from toxic endophyte tall fescue can increase and stabilize in less than 2 wk, but alkaloid-induced vasoconstriction that causes a vulnerability to severe heat stress is not alleviated within 30 d.

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.

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.