Recipient Organization
UNIVERSITY OF NEVADA
(N/A)
RENO,NV 89557
Performing Department
Ag Nutrition and Vet Sciences
Non Technical Summary
Winter season standing forage for grazing animals both wild and domestic in the Great Basin is often in short supply, and sometimes limited in species composition. This is particularly true in several of the valleys of south-central Nevada. In some areas, one or two shrub species comprise 75% or more of the available forage. Dry Lake Valley located north and west of Pioche, NV, is one of these valley types. The primary forage species are black sagebrush (Artemisia nova) on the valley shoulders, and Nevada ephedra (Ephedra nevadensis) in the valley bottom. Another species of ephedra, green ephedra (Ephedra viridis) also occurs in the more upland areas within the sagebrush matrix. Ephedra contains ephedrine, a central nervous system stimulant. High consumption rates of Nevada ephedra may be responsible for major sheep mortality events in the area. Mortality symptoms indicate a central nervous system malfunction. We propose to: 1) Determine the amount and types of ephedrine contained and in the two species of ephedra growing in the area of interest; 2) Determine the nutritional value (crude protein and total digestible nutrients) of the two species using chemical analysis; and 3) Determine the rumen fermentation characteristics (volatile fatty acids) of the two species using the dual flow continuous culture system and in vitro techniques. Once these content profiles are determined, determine a management prescription to minimize sheep mortality that has been ongoing for decades in the area. In one case, an operator lost over 200 ewes. Very high mortality rates reduce income for sheep operators, leading to reduced economic turnover in local economies of rural Nevada.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
The primary goal of this study is to assist Nevada ranchers (especially sheep operations) in dealing with a rangeland management problem associated with domestic livestock foraging on Ephedra species. In order to achieve this goal, we will address these specific objectives: 1) Investigate the nutritive content of Ephedra species by chemical analyses (neutral detergent, acid detergent fiber, organic matter, and crude protein); 2) Investigate ephedra test diet rumen fermentation products (short chain fatty acid content, pH, ammonia nitrogen content, and organic matter digestibility) using a dual flow continuous culture system from test diets of: a 100% diet of native dormant grass (control); a 50-50% diet of E. viridis and dormant native grass; and a 50-50% diet of E. nevadensis and dormant native grass; and 3) Quantify alkaloid content (ephedrine) of both ephedra species using chromatography.
Project Methods
Overall designThe basic study design will be two-way analysis of variance that includes three time periods (December, January, February) and two species of ephedra as fixed treatments. Ephedrine content and composition, tannin content, and nutritional content will be analyzed with this design. The second design will be a two-way analysis of variance assessing the volatile fatty acid compositions of the two ephedra species along with three additional test diets. Dual flow continuous culture techniqueA total of six fermenters will be used as a closet in vitro model to simulate ruminal microbial fermentation (Hoover et al. 1976; Hannah et al. 1986). The three treatment diets: 1) 100% grass hay; 2) 50-50% green ephedra and grass hay; and 3) 50-50% Nevada ephedra and native grass hay, will be run in duplicates. Rumen fluid will be collected from rumen canulated steers at the University of Nevada-Reno Main Station Farm. Immediately the rumen fluid will be strained through four layers of cheese cloth into a pre-warmed, insulated thermos. Upon arrival at the UNR nutrition laboratory rumen fluid will be combined and bubbled with CO2 gas. One liter of rumen fluid will be used for each fermenter. The fermenters are equipped with an automated feeding system. Mineral buffer solution (Weller and Pilgrim 1974) will be infused into each fermenter at the rate of 1.5 ml/min. to obtain a liquid dilution rate of 0.10 h-1 and a solid (overflow) dilution rate of 0.05 h-1. A pH of 6.8 ± 0.05 will be maintained by automatic infusion of 3 N HCl or 5 N NaOH and regulated by a pH controller. Anaerobic conditions will be achieved by continuous infusion of nitrogen gas at rate of 40 ml/min. Temperature (39oC) and mixing of fermenters are controlled with VirTis Omni-culture base units (The VirTis Company Gardiner, NY). Each fermenter will be supplied daily with 75 g dry matter, ground (2 mm screen) of test diet by an automated feeding mechanism adjusted to deliver the forage in 12 equal portions over a 24 h period.Chemical analysis: Nutrient content Absolute dry matter will be determined on freeze dried digesta and forage test diets by drying at 105 oC for 24 hours and will be followed by ashing at 500oC for 16 h in a muffle furnace to determine organic matter. The nitrogen content (AOAC 2000) and acid detergent and neutral detergent fiber (Jeraci et al. 1988) in digesta and test diets will be determined.Effluent samples from continuous cultures will be prepared (Erwin et al.1961) for short chain fatty acids analysis and their concentration will be determined by gas chromatography (Varian Model 13800, Varian Inc. Walnut Creek, CA) equipped with a glass column (180 cm x 4 mm i.d.) packed with GP 10 % SP-1200/ 1% H3PO4 (Supelco, Bellefonte, PA). Helium will be used as a carrier gas with a flow rate of 85 ml/minute. The oven, injection port, oven and detector (FID) port temperatures will be 125, 175, and 180oC, respectively. Ammonia nitrogen in the effluent will be measured calorimetrically (Chaney and Marbach 1962). Digestibility of DM, OM, NDF and CP will be calculated according to Hannah and Stern (1985). The data will be analyzed by using the GLM procedures of SAS (SAS Inst. Inc. Cary, NC).Alkaloid content determination Since ephedrine is a regulated by the Drug Enforcement Administration as a controlled substance, alkaloid content and identification will be conducted outside UNR by a DEA certified contract laboratory. The method described in this procedure is simple, sensitive, accurate and rapid for the simultaneous identification and determination of all six alkaloids (Jian-fang Cui et al. 1991).Five grams of dried ephedra plants will be weighed into a 10 ml centrifuge tube and 2 ml of 0.65 M KOH solution, 1.2 g of NaCl and 2.0 ml of diethyl ether containing 10 ppm diphenylamine will be added to the tube. The mixture will be shaken for 15 min then centrifuged for 7 min at 1600 x g. The organic layer will be pipetted into a 2 ml sample vial containing 0.1 g of anhydrous Na2SO4 and the extract solution (2µL) will be submitted to GC/NPD analysis. The concentrations of the six alkaloids in ephedra will be calculated according to the regression equations that will be deduced using appropriate standards.Alkaloid content identification An aliquot (25 mg) of the powdered drug will be weighed and transferred to a 10 ml centrifuge tube, mix with 3 ml of 0.5 M H2SO4 solution. The mixture will be shaken for 15 min. and centrifuged for 7 min. at 1600 x g. The supernatant layer (2 ml) will be transferred to another tube, and add 0.7 ml of 5 M KOH solution, 1.2 g NaCl and 2 ml of redistilled diethyl ether. The mixture will be shaken for 15 min and centrifuged at 1600 x g for 7 min. The ether layer will be taken into a 2 ml sample vial and evaporate to dryness at room temperature under a slow stream of nitrogen. Then 100 µL of MSTFA will be added to the residue. The mixture will be reacted at 75oC for 15 min, and submit 1 µL of the resulting solution to GC analysis. This is to confirm the occurrence of all six alkaloids by comparison of their GC/MS data with those of the corresponding standards. After the alkaloid content determination is complete, we willestimate the rate and amount of ephedrine actually consumed on a per head basis. This will provide us with an estimate of how much ephedra will be necessary to conduct an animal feeding trial as a follow-up to this initial study.