5.3. Forage Analysis

Grab samples of forage were collected during the time period when cattle were grazing the endophyte treatments (late gestation to early lactation) from each paddock when cattle were moved to a new strip of grazing and hay samples collected before feeding. All forage and hay samples were freeze dried and ground using a 1-mm screen Wiley cutting mill (Arthur H. Thomas, Philadelphia, PA, USA). Forage samples were composited on a monthly basis. In year 1, forages and hay were analyzed for total ergot alkaloid infection concentrations (Agrinostics Limited Co., Watkinsville, GA, USA). As ergovaline and its epimer ergovalinine are the most informative ergots involved in vasoconstriction [5,8], we developed an assay to measure concentration of these ergopeptines to obtain more specific information. This assay was not available in year 1 but was developed and utilized in year 2 of this experiment. In year 2, forage and hay samples were analyzed for ergovaline and ergovalinine concentrations by LC-MS/MS at the Multi-User Analytical Laboratory (MUAL) at Clemson University.

For quantification of ergovaline and ergovalinine concentrations, freeze dried forage and hay samples were extracted according to Guo et al. [65]. A tall fescue seed extract was provided by J. L. Klotz and used to establish a calibration range from 0.78 to 100 ng/mL to quantify ergovaline and ergovalinine [66]. Ergotamine tartrate (Sigma Chemical Co., St. Louis, MO, USA) was used as an internal standard. Samples were analyzed using a Thermo Orbitrap Fusion™ Tribrid™ Mass Spectrometer (Thermo Fisher, Waltham, MA, USA) with MS1 (ESI positive, 60,000 resolution, scan range 220–1200 m/z) and MS2 (HCD & CID on targeted mass of 25 ergot alkaloids in orbitrap at 15,000 resolution) in the Clemson University Multi-User Analytical Laboratory and Metabolomics Core. The column was Phenomenex Kinetex XB-C18 with 1.7 μm, 100A, 150 × 2.1 mm (Phenomenex, Torrance, CA, USA). Solvent A was 5 mM ammonium bicarbonate and solvent B was 90% acetonitrile with 5 mM ammonium bicarbonate. Quality control checks were performed using instrument blanks, method blanks to determine recovery, and instrument quality control to assess relative standard deviation of 12.5 ppb ergovaline standard. The percent recovery for ergovaline, ergovalinine, and ergotamine were 98, 95, and 100%, respectively. Compound detection was performed using the extracted ion chromatogram for targeted analytes within 2 ppm mass error in Skyline software. Quantification of ergovaline and ergovalinine was performed using linear external calibration curves (0.78 to 100 ng/mL. The method limit of detection for ergovaline/ergovalinine in grass and hay was 11.414 ng per g freeze-dried plant tissue (instrument LOD of 0.78 ppb).