Endophyte infected tall fescue (E+) is the base diet for nearly all beef cattle in the southern USA. It has been linked to a variety of toxicological conditions due to the presence of large numbers of ergot alkaloids. This study was designed to investigate the effects of E+ seed extract and selected ergot alkaloids on the detoxification pathway by cytochrome P450 (CYP3A4) enzyme system. Tests were performed using the P450- Glo CYP3A4 enzyme activity kit (Promega, WI), according to the manufacturer’s manual. Luminescence was measured using a single tube TD20/20 luminometer. Endophyte infected tall fescue seed was extracted with 50/50 methanol/25 mM ammonium carbonate, cleaned and concentrated on Strata-X reversed phase column (Phenomenex). The extracts were evaluated on an HPLC, and then tested using a serial dilution method. Commercially available ergonovine (EN), ergocorine (ER), bromocryptine (BC) and ergocryptine (EC) were tested individually using 0 to 44 nM concentrations. Seed extract of E+ produced a significant (P P < 0.05) dose dependent manner with EC being most potent, followed by ER, BC, and then EN (70%, 40%, 30% and 10% at 44 nM concentration). The similarity of the inhibition curves of seed extract to that of the commercially available ergot alkaloids suggests a related mode of action and that the use of such ergot alkaloids and CYP3A4 assay is a good model to study the toxicity of tall fescue. Furthermore, it provides the foundation to identify the individual toxic components of purified endophyte infected tall fescue extract.

This study was conducted to investigate the effect of the ergot alkaloid, ergotamine (ET), on the induction of CYP3A and the interaction in vivo and in vitro with ET.Sprague-Dawley rats were treated intraperitoneally for 4 days as follows: control (injecting with 0.5 ml of only corn oil); dexamethasone treatment (injecting with 100 mg/kg of dexamethasone in corn oil); and ergotamine treatment (injecting with 100 mg/kg of ergotamine in corn oil).Liver tissues were collected from each group (n = 5, total of 30 rats) and liver microsomes were prepared.Cytochrome CYP3A activity was evaluated using ET and its isomer as substrates in medium containing liver microsomes and NADPH at 37˚C for 30 min.HPLC was used to measure the disappearance of the substrate and the appearance of the metabolites.Liver microsomes from rats pretreated with dexamethasone were five times more (P < 0.01) active than microsomes from the control animals in the biotransformation of ET (32.1 and 7.0 nM/min/mg protein, respectively; SE = 4.83) or ET-isomer (21.6 and 4.7 nM/min/mg protein, respectively; SE = 1.7079) into its corresponding ET metabolites.The ergotamine treatment produced no increase (P > 0.05) in activity of CYP3A when compared to the control group (5.2 vs 7.0 nM ET/min/mg protein; SE = 4.83) or ET isomer (1.5 vs 4.7 nM ET isomer/min/mg protein; SE = 1.70).When ketoconazole was used as specific inhibitor of CYP3A, ergotamine metabolisms were inhibited in a dose dependent fashion reaching a maximum at an inhibitor to substrate ratio of greater than one and LD50 at 0.5 nM of ketoconazole/mg protein.The data presented in this study suggest that although the ergot alkaloids ergotamine and its isomer are ideal substrates for the isozyme CYP3A, these compounds have no effect on the induction of CYP3A after 4 days of treatment.

There is evidence that ergot alkaloids can directly interact with mammalian spermatozoa affecting sperm functions. Ergot alkaloids exert their toxic or pharmaceutical effects through membrane receptor-mediated activities. This study investigated the signaling pathways involved in the in vitro inhibitory effects of both ergotamine (ET) and dihydroergotamine (DEHT) on the relative motility of bovine spermatozoa using specific inhibitors. Motile bovine spermatozoa were prepared using a Percoll gradient and incubated with ergot alkaloids with and without signaling pathway inhibitors. Co-incubation of ET or DHET with 100 μM prazosin (alpha 1-adrenergic receptor inhibitor) decreased (p < 0.05) relative motility of spermatozoa when compared with controls. In addition, preincubation of spermatozoa with 10 or 20 μM prazosin and DHET also reduced (p < 0.05) the number of motile spermatozoa. Relative sperm motility (motility of treated spermatozoa normalized to control sperm motility) was increased (p < 0.05) when co-incubations included ET and yohimbine (alpha 2-adrenergic receptor inhibitor); conversely, co-incubation of yohimbine (100 μM) and DHET decreased (p < 0.05) the percentage of motile spermatozoa when compared with controls. Pertussis toxin and cholera toxin (effectors of inhibitory and stimulatory G-proteins, respectively) altered (p < 0.05) relative sperm motility in a concentration dependent manner; however, co-incubation of pertussis or cholera toxin with ergot alkaloids had no interactive (p = 0.83) effects on the relative motility of spermatozoa. Co-incubation of Rp-cAMP (a membrane-permeable cAMP inhibitor) with 50 μM DHET had no effect (p > 0.05) on relative sperm motility; whereas, the co-incubation of 22.4 or 44.8 μM Rp-cAMP with 50 μM ET increased (p < 0.05) the percentage of motile spermatozoa when compared with 0 or 224 μM Rp-cAMP (49%, 65%, 59%, and 54%, respectively, for 0, 22.4, 44.8, and 224 μM of Rp-cAMP. An interaction between BAPTA-AM (a chelator of intracellular calcium) and alkaloids also impacted (p < 0.05) relative sperm motility. Generally, co-incubating spermatozoa with BAPTA-AM and ET increased the percentage of motile spermatozoa; however, co-incubation with DHET decreased relative sperm motility except with 41 μM BAPTA-AM. Collectively, these observations suggest that ET and DHET decreased the percentage of motile bovine spermatozoa via alpha adrenergic receptors. However, the second messenger systems involved with ergot alkaloid inhibition of relative motility of bovine spermatozoa remain to be elucidated.

ABSTRACT Beef steers representing four biological types were evaluated for fatty acid profiles and meat characteristics in muscle tissue developed on grazed forages. Biological types included six large‐framed, late maturing, nine medium‐framed, late maturing, nine medium‐framed, intermediate maturing, and nine medium‐framed, early maturing steers. Longissimus dorsi muscles were removed and frozen after 7 days of dry aging. Tenderness and cook loss were similar between biological types. Significant differences ( P < 0.05) in fatty acid profiles were found between biological types. The medium‐framed, early maturing animals scored highest ( P < 0.05) in marbling, had the highest ( P < 0.05) concentration of total n‐3 fatty acids, and had the lowest n–6/n–3 ratio at 1.03:1. All biological types had a mean n–6/n–3 ratio well below the recommended < 4:1 for human cardiovascular health. Under this dietary management system, medium‐framed, early maturing beef steers should be considered for producing grass‐fed beef based on both carcass merit and fatty acid composition. PRACTICAL APPLICATIONS Beef cattle that reach physiological maturity (i.e. puberty) earlier than their contemporaries should be selected for the production of grassfed beef if fully developed on forages. Those animals have the ability to deposit fat earlier than their later‐maturing contemporaries. Additionally, medium‐framed beef animals should be selected over larger‐framed animals as skeletal frame does not contribute to meat quality but requires more nutritional input for growth and maintenance. This ability to deposit fat exclusively on forage nutrients, without energy or protein supplementation, should be exploited when developing grassfed beef. Fat deposition, particularly intramuscularly (IM), contributes positively to meat quality. Fatty acids, especially the ratio of n‐6/n‐3, are optimized when beef animals have been fully developed on nutrient‐dense forages. Forages must be managed in such a way to capture those nutrients for animal intake and protect the plants for regrowth in a pasture setting.

Summary Much of the research on fescue toxicosis has concentrated on evaluating animal response to grazing endophyte-infected (E+) versus endophyte-free tall fescue or the effects of single toxins such as ergonovine (EN), ergovaline (EV), or ergotamine (ET) on animal performance. Such approaches have eliminated the opportunity to test the possible additive, synergistic, or antagonistic interactions of one or more ergot alkaloids with the other ergot alkaloids found in E+ tall fescue. This study was conducted to determine the effects of simultaneous exposure of pairs of EN, EV, and ET on the kidney adenosine triphosphatase (ATPase) system in vitro. Tests were performed using three separate rat kidney homogenates and were repeated four times at concentrations of 0, 75, and 200 μ M . Individually, EN, EV, and ET induced dose-dependent inhibitions of kidney Na + /K + ATPase, with EN being most potent, followed by purified EV, and then by ET. The ergot alkaloids inhibited Mg 2+ ATPase to a lesser degree than Na + /K + ATPase, with EN again being the most potent toxin. Simultaneous exposure to any combination of the ergot alkaloid pairs tested (EV+ET, EV+EN, and ET+EN) resulted in significant interactions ( P <0.05), indicating antagonistic effects on the inhibition of Na + /K + ATPase and Mg 2+ ATPase for most concentration combinations. These interactions suggest that in studies of the effects of any ergot alkaloid on animal performance, effects of other ergot alkaloids may also be present. Effects may not be additive, as was the case in this study, and the presence of one toxin may enhance or hinder the effectiveness of others.

A simple, rapid method of liquid solid extraction coupled with a sensitive HPLC procedure with fluorescence detection to measure ergot alkaloid levels in bovine blood samples is presented. Single injections of either ergosine, ergotamine, or ergine were given to Holstein steer calves (140 kg), and serum concentrations of the ergot alkaloids were monitored over a 2-h period. Serum samples were loaded on a Bond Elut (100 mg) C-18 column that had been washed with methanol and distilled water. Ergot alkaloids were then eluted with methanol and analyzed by HPLC. Recovery of ergosine, when added to serum at concentrations of 0.5-24.0 ng/mL, ranged from 95 to 98%. The data fit previous reports of three phases in elimination of drug. The method reported has excellent repeatability, a detection limit of 0.5 ng/mL, and provides an alternative to quantitate ergosine, ergotamine, and ergine in bovine blood samples at therapeutic dosages.