Bovine neuronal vesicular glutamate transporter activity is inhibited by ergovaline and other ergopeptines.

l-Glutamate (Glu) is a major excitatory neurotransmitter responsible for neurotransmission in the vertebrate central nervous system. Vesicular Glu transporters VGLUT1 and VGLUT2 concentrate (50mM) Glu [Michaelis constant (measuring affinity), or K(m),=1 to 4mM] into synaptic vesicles (SV) for subsequent release into the synaptic cleft of glutamatergic neurons. Vesicular Glu transporter activity is dependent on vacuolar H(+)-ATPase function. Previous research has shown that ergopeptines contained in endophyte-infected tall fescue interact with dopaminergic and serotoninergic receptors, thereby affecting physiology regulated by these neuron types. To test the hypothesis that ergopeptine alkaloids inhibit VGLUT activity of bovine cerebral SV, SV were isolated from cerebral tissue of Angus-cross steers that were naive to ergot alkaloids. Immunoblot analysis validated the enrichment of VGLUT1, VGLUT2, synaptophysin 1, and vacuolar H(+)-ATPase in purified SV. Glutamate uptake assays demonstrated the dependence of SV VGLUT-like activity on the presence of ATP, H(+)-gradients, and H(+)-ATPase function. The effect of ergopeptines on VGLUT activity was evaluated by ANOVA. Inhibitory competition (IC(50)) experiments revealed that VGLUT-mediated Glu uptake (n=9) was inhibited by ergopeptine alkaloids: bromocriptine (2.83±0.59µM)

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.

The bull sperm microRNAome and the effect of fescue toxicosis on sperm microRNA expression.

Tall fescue [Schedonorus phoenix (Scop.) Holub] accounts for nearly 16 million hectares of pasture in the Southeastern and Mid-Atlantic U.S. due to its heat, drought, and pest resistance, conferred to the plant by its symbiotic relationship with the endophyte Neotyphodium coenophialum. The endophyte produces ergot alkaloids that have negative effects on the growth and reproduction of animals, resulting in the syndrome known as fescue toxicosis. The objectives of our study were to identify microRNA (miRNA) present in bovine sperm and to evaluate the effects of fescue toxicosis on sperm miRNA expression. Angus bulls were assigned to treatments of either toxic or non-toxic fescue seed diets. Semen was collected and subjected to RNA isolation. Three samples from each treatment group were chosen and pooled for deep sequencing. To compare miRNA expression between treatment groups, a microarray was designed and conducted. For each of the top ten expressed miRNA, target prediction analysis was conducted using TargetScan. Gene ontology enrichment was assessed using the Database for Annotation, Visualization and Integrated Discovery. Sequencing results elucidated the presence of 1,582 unique small RNA present in sperm. Of those sequences, 382 were known Bos taurus miRNA, 22 were known but novel to Bos taurus, and 816 were predicted candidate miRNA that did not map to any currently reported miRNA. Of the sequences chosen for microarray, twenty-two showed significant differential expression between treatment groups. Gene pathways of interest included: regulation of transcription, embryonic development (including blastocyst formation), Wnt and Hedgehog signaling, oocyte meiosis, and kinase and phosphatase activity. MicroRNA present in mature sperm appears to not only be left over from spermatogenic processes, but may actually serve important regulatory roles in fertilization and early developmental processes. Further, our results indicate the possibility that environmental changes may impact the expression of specific miRNA.