2008 Annual Report 1a.Objectives (from AD-416) Objective I: Pine Needles 1.1 Determine if isocupressic acid (ICA; the abortifacient compound in pine needles) concentration in pine needles is modulated by the environment. 1.2 Identify the matabolites of isocupressic acid in pine needles that cause abortions in cattle. Determine the biological mechanism, develop diagnostic techniques, and therapeutic procedures. 1.3 Determine factors that influence cattle consumption of pine needles and develop management practices to prevent abortion. Objective II: Broom Snakeweed 2.1 Identify the toxic and abortifacient compounds in broom snakeweed. 2.2 Describe the ecology of broom snakeweed, develop management and control guidelines to reduce incidence of poisoning and abortion in livestock. Objective III: Lupine 3.1 Isolate, identify, and evaluate toxicity and teratogenicity of lupine alkaloids which cause birth defects in calves born to cows that graze these plants. 3.2 Evaluate the role of genotype and environment on lupine alkaloids, and thus the relative toxicity of various species and populations of lupine. 3.3 Determine the physiological mechanism of lupine-induced birth defects and evaluate the maternal and fetal toxicokinetics of alkaloids. 3.4 Evaluate the influence of climate on population cycles of lupine. 3.5 Determine the importance of lupines as nutritional components for cattle during critical times of the year. 3.6 Identify conditions under which cattle graze various lupine species. Objective IV: Veratrum 4.1 Develop models to study the toxicokinetics, including clearance times, and toxicity of steroidal alkaloids in Veratrum californicum. 1b.Approach (from AD-416) 1.1 Data on environmental conditions will be collected at each site using local weather stations. ICA levels and environmental conditions will be correlated to determine if any patterns emerge. Soil samples will be collected at each site for future evaluation. 1.2 Samples of maternal and fetal tissues will be collected for histologic analysis and determination of ICA concentrations using existing ELISA’s and GC/MS methods. Proteomic analyses via LC/MS/MS techniques will be done. 1.3 Pen and field studies using cattle in high, medium and low body condition will be done to determine effects on needle consumption and grazing times. Nutrient supplements will be offered to determine if pine needle consumption will be altered. 2.1 The diterpene acid “fingerprint” of broom snakeweed from various populations in Arizona, New Mexico and Utah will be determined by chemical analysis. Subsequent in vitro and in vivo studies will be done to determine abortifacient activity. 2.2 A grazing study will be conducted to determine if various management practices can be implemented to force cattle to graze snakeweed as a biological control. A clipping study will be conducted to further describe the effects of defoliation on snakeweed and the surrounding plant community. 3.1 Alkaloids will be isolated by chemical methods and identified by chromatography, NMR, mass spectrometry, and elemental analysis. Toxicology will be evaluated using a mouse bioassay and cell lines that express nicotinic acetylcholine receptors. 3.2 A chemical fingerprint of Lupinus sulphureus collected from different locations will be generated using chemical methods. Fingerprints will be analyzed via cluster analysis and phylogenetic analysis will be performed using AFLPs (Amplified Fragment Length Polymorphisms) to determine the genetic relationship of the populations. 3.3 Pregnant goats in late gestation will be used to determine the rate of absorption, distribution and elimination of the teratogenic alkaloids. The pharmacokinetic profiles of the alkaloids will be compared between maternal and fetal systems. 3.4 Established transects will be monitored over the next 5 years to determine the influence of weather patterns on lupine density. Correlations of lupine age, class, density, and trends will be made with seasonal precipitation and temperature. 3.5 Consumption of lupines by cattle on rangelands dominated by low quality forages may be related to nutrient content. Twelve yearling heifers in a field study will be supplemented with different levels of protein to compare lupine ingestion. 3.6 Short-duration and high intensity grazing studies in early, mid, and late summer will be used to determine what role grazing pressure has on lupine intake during different seasons of the year. 4.1 A monogastric model (swine) will be used to determine the kinetics (clearance and metabolism) of a well known teratogenic alkaloid from Veratrum. This pilot project will be a model for testing the clearance of other plant toxins from animal tissues to evaluate food safety of animal products. Clearance rates between the monogastric model and small ruminant model will be compared. 3.Progress Report This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P. In pen studies, cattle showed preference to different lupines containing different profiles of alkaloids. From this study, six different lupine plants growing in the same region were ranked ordered in terms of cattle preference. Lupine seasonal grazing studies showed that cattle did not prefer lupine early in the growing season, but were forced to graze it as other forage became limited. However, lupine was preferred forage late in the growing season when other forage was dry and dormant. Enantiomeric alkaloids that cause birth defects (teratogens) were isolated and their toxicities determined in both mouse and cell line bioassays. The enanatiomeric pairs were found to have the same relative toxicities in both the mouse and cell line assays. These alkaloids were used in a pilot study in which the alkaloids were administered to the fetus via osmotic mini-pumps. Preliminary data on the maternal-fetal transfer of teratogenic alkaloids indicates the fetus circulation profile mimics the material profile but is delayed. Utah Juniper bark and needles containing primarily agathic acid as the diterpene acid were dosed to cattle. The cow serum metabolites of agathic acid were the same as the cow serum metabolites as isocupressic acid, the primary abortifacient in Ponderosa Pine needles. In addition, pregnant cows that were dosed with Utah Juniper aborted. This study determined agathic acid is an abortifacient. Vegetative response to defoliation showed snakeweed mortality increased, blue bunch wheat grass cover declined, and crested wheat grass increased. In order to better understand the mechanism of action of isocupressic-induced abortions, an ELISA was used to determine the distribution of isocupressic acid and its metabolites in cows. Veratrum californicum has been shown to be teratogenic in sheep. A detailed analysis of the kinetic profile of cyclopamine in sheep was initiated. 4.Accomplishments 1. Effect of cow body condition on consumption of pine needles (PN). Pregnant cattle that eat pine needles (PN) often abort. There is little information on the potential effects of body condition on PN consumption by cattle. We determined if cows in low (LBC) or high body condition (HBC) would consume different amounts of green pine needles (Pinus ponderosa). Mean BCS (1 = emaciated, 9 = obese) was 7.5 for HBC and < 4.0 for LBC cows during the experiments. During Exp. 1, LBC cows consumed more pine needles than did HBC cows. During Exp. 2, LBC cows consumed variable, but greater amounts of pine needles than did HBC cows. When fed a high protein/low energy diet, LBC cows ate more pine needles than did HBC cows. When fed a low protein/high energy diet, LBC cows consumed more pine needles than HBC cows for the first 3 d of the study, and then consumption by LBC animals decreased during the last 4 d. These experiments indicate the dietary protein/energy ratio may be an important factor in the ability of cows to tolerate PN terpenes, as cows were not able to sustain high levels of needle consumption on a low protein diet. During the 25-d grazing study, LBC animals selected more pine needles (up to 25% of daily bites) on some days compared to HBC cows. Weather influenced pine needle consumption as pine needle bites by LBC cows were related to days of greater snow depth, and lower minimum daily temperatures. Both LBC and HBC cows increased selection of pine needles from trees during cold, snowy weather, but the magnitude of the increase was greater for LBC cows. These results indicate livestock producers can reduce consumption of pine needles and risk of abortions by pregnant cows by keeping cows in moderate body condition. This research relates to NP 215 Rangeland, Pasture and Forage, Component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P. 2. Blood Serum metabolites in cattle dosed Utah Juniper bark and needles containing agathic acid. Pregnant cattle that eat pine needles (PN) often abort. There is little information on the abortifacient activity of metabolites of isocupressic acid. Pregnant cattle were dosed with Utah juniper bark and needles which contain agathic acid as the major diterpene acid. Agathic acid is one of the major serum metabolites observed in serum of cattle dosed with the abortifacient compound isocupressic acid. All three cows dosed with Utah juniper bark containing agathic acid aborted their calves after 4 – 6 days of treatment. A fourth cow was dosed with Utah juniper needles which contained lower levels of agathic acid. This cow developed complications after 13 days and the calf was pulled. Serum was taken from all cows and analyzed for known metablolites of isocupressic acid. The metabolites identified included agathic acid, dehydroagathic acid, and tetrahydroagathic acid. These are the same metabolites observed in serum of cattle dosed with Ponderosa pine needles or isocupressic acid. The rate of abortion after dosing was correlated to serum metabolites concentrations in the blood. These experiments implicate agathic acid as an abortifacient compound in cattle. Provides new information on the abortifacient properties of labdane acids in cattle. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P. 3. Perform pilot study to test feasibility of using an ELISA for studying the tissue distribution of isocupressic acid (ICA) and its metabolites. Pine needles induce abortion in cattle presumably by restricting blood flow to the fetus. Although ICA has been shown to be the compound in pine needles responsible for inducing abortions, ICA is not a vasoactive compound. Therefore the mechanism by which ICA causes a restriction in blood flow to the fetus remains unknown. In order to better understand the mechanism of action of ICA-induced abortions, the tissue distribution of ICA and its structurally similar metabolites were determined using an ELISA developed for the detection of ICA. After treating cattle with ICA for two days the animals were necropsied and all tissues were analyzed for ICA content. This pilot study was successful in that ICA was detected in cattle serum, urine, and tissues using the ELISA. The characterization of ICA distribution in cattle will aid in understanding the mechanism of action of pine needle abortion. Once this mechanism is fully understood then potential therapies or management strategies can be developed and/or refined in order to reduce losses. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P. 4. Plant community response to snakeweed grazing and defoliation. Broom snakeweed prevents optimum utilization of rangelands in the western United States. Grazing trials were conducted in spring and late summer to determine if cattle could be induced to graze snakeweed as a biological control. The cows grazed 62 to 95% of snakeweed plants and removed 50 to 85% of its biomass. Snakeweed density declined by 50% compared to ungrazed areas. In spite of the high grazing pressure, crested wheatgrass was maintained in the spring-grazed pastures and increased in the summer-grazed pastures. In a companion clipping trial, snakeweed mortality increased when it was clipped by itself, and along will all other vegetation simulating intensive grazing. Crested wheatgrass recovered following clipping and provided competition which further reduced snakeweed. Bluebunch wheatgrass declined following clipping and was not competitive with snakeweed. Defoliation in spring of healthy stands of both crested wheatgrass and bluebunch wheatgrass may allow snakeweed seedlings to establish. This is new information showing broom snakeweed can establish in healthy plant communities. This information will be used by land managers and ranchers in maintaining healthy rangelands. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P. 5. Effect of lupine alkaloids on livestock preference for lupine species. Intake of lupine by livestock is variable and difficult to predict. Management of lupine species would be greatly aided by understanding factors influencing consumption, including the effect of the alkaloid composition on livestock intake of lupine. A pen study was conducted to relate the alkaloid composition of 6 different lupine species and consumption of fresh lupine by cattle. In rank order cattle preferred L. leucophyllus (Washington); L. polyphyllus; L. sulphureus (Oregon); L. leucophyllus (Oregon); L. sulphureus (Washington); L. sericeus (Washington). This study is ongoing; fresh and dried lupines, and various alkaloid extracts, will be compared in future studies. There is a critical need to understand how alkaloid concentrations impact cattle ingestion of lupine. A clearer understanding of the role of alkaloids in mediating interactions of grazing cattle and lupines will provide useful information for manipulating dietary preferences to reduce cattle losses from lupines. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P. 6. Isolate, identify, and evaluate toxicity and teratogenicity of lupine alkaloids which cause birth defects in calves born to cows that graze these plants. The functional parameters of potency and efficacy of plant toxins in two cell lines that express neuromuscular or ganglionic nicotinic acetylcholine receptors is not known nor how they correlate with toxicity in the mouse. Currently, we know coniine and N-methylconiine in poison hemlock and anabasine and N-methylanabasine in wild and domestic tobacco are teratogenic. We also have evidence implicating ammodendrine and N-methyl ammodendrine present in Lupine spp. as teratogens. However, these alkaloids occur in plants as racemic mixtures that contain both the + and – forms of these compounds. There is little information available on the relative toxicity + and – forms of these compounds. The culture of the two cell lines initiated, membrane potential fluorescence assays were optimized, and initial screening of the alkaloids was started. The toxicity of the enantiomers of coniine was determined in mice and a cell line that expresses neuromuscular type nicotinic acetylcholine receptors. Toxin/chemical enantiomers were determined to have different toxicities from each other in both mice and the cell line assay. Significantly, toxin/chemicals had the same order of toxicity in both the mice and the cell line assay. These results show that the enantiomers of toxins in plants must be identified and the toxicity of both enantiomers determined to fully understand the toxicity of the toxin and the plant. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P. 7. Influence of body condition on snakeweed consumption. Broom snakeweed increases and dominates rangelands following disturbances such as overgrazing, fire, and drought. It is also poisonous, causing abortions in cattle. Even though overgrazing is a principal cause of its increase, prescribed grazing may be effective in controlling it. Grazing trials were conducted in 2004 (body condition) 2005 (nutrient supplement), 2006 (grazing pressure), and 2007 (body condition) to evaluate their efficacy in enhancing cattle to graze snakeweed. Cattle in low body condition grazed more snakeweed. Grazing pressure was the major factor influencing cattle to graze snakeweed. Restricting forage allowance to 50% or less of daily intake requirement caused cows to graze 62 to 95% of snakeweed plants and remove 50 to 85% of snakeweed biomass. Cows maintained weight and body condition during the studies, and there was no sign of toxicity. In this study grazing pressure was identified as the major factor inducing cattle to graze broom snakeweed and invasive weeds in general. This principle and grazing protocol can be used to induce livestock to graze invasive weeds as a biological control. Intensive grazing of weeds can be used in plant communities seeded to grazing-resistant species, but likely not in native plant communities that will degrade under heavy grazing. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P. 8. Characterization of kinetic profile of cyclopamine in sheep. The plant Veratrum californicum is known to be teratogenic to sheep causing a number of birth defects dependant upon the time of exposure, with a cyclopic lamb formation being the most noted. Although cyclopamine has been shown to be the teratogenic compound a detailed analysis of the kinetic profile of cyclopamine in sheep has not been performed. A detailed characterization of the kinetic profile of cyclopamine in sheep was begun. Five sheep were dosed orally with ground Veratrum californicum plant material and blood was collected over a 72 hr period to analyze for cyclopamine concentration. Additionally, five sheep were dosed intravenously with purified cyclopamine and blood collected and analyzed for cyclopamine concentration. These studies were performed to understand the kinetic profiles of cyclopamine in sheep prior to performing similar studies in a monogastric animals, such as pigs. The characterization of the toxicity, teratogenicity, and kinetic profile of cyclopamine in a monogastric animal, such as a pig will provide new information on kinetics in non-ruminants. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P. 9. There are numerous alkaloids in plants that cause birth defects. There are numerous teratogenic alkaloids in plants. Some of which are produced as enantiomeric pairs. Information is not available to describe the variation in toxicity or teratogenicity of the individual enantiomers. Teratogenic alkaloid and their stereoisomers have been tested for toxicity in mice. A pilot study in pregnant goats was initiated using coniine HCL administered to the fetus via osmotic mini-pumps. This information will expand the knowledge base about teratogenic alkaloids and provide data to better assess risk of grazing pastures infested with these plants. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P. 10. Lupines cause heavy losses to the cattle industry. Lupines induce "crooked calf disease" in the north western U.S. Currently, there is no information on absorption, distribution, and elimination of the teratogenic alkaloids from the fetus or the comparison to that of the maternal kinetics. Preliminary data indicates anabasine directly enters the fetal circulation and the absorption profile mimics the maternal profile. However, the elimination profile appears to be delayed in the fetus compared to the maternal elimination rate. This research will provide new information on the fetal kinetics of teratogenic alkaloids. This will improve the level of understanding for scientists and ranchers on the risk of grazing lupine pastures. The results of this research have biomedical implications. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of New Germplasm Releases 1 Number of Web Sites Managed 10 Number of Other Technology Transfer 4 Review Publications Lee, S.T., Cook, D., Panter, K.E., Gardner, D.R., Ralphs, M.H., Motteram, E.S., Pfister, J.A., Gay, C. 2007. Lupine induced "Crooked Calf Disease" in Washington and Oregon: Identification of the alkaloid profiles in Lupinus sulphureus, Lupinus leucophyllus, and Lupinus sericeus. Journal of Agricultural and Food Chemistry, Web release 11/27/2007 Article DOI: 10.1021/jf0723110 Journal releae Volume 55, Number 26, pp. 10649-10655. Panter, K.E., Gardner, D.R., Lee, S.T., Pfister, J.A., Ralphs, M.H., Stegelmeier, B.L., James, L.F. 2007. Important poisonous plants of the United States. Veterinary Toxicology: Basic and Clinical Principles, Chpt. 66 pp. 825-872. Weinzweig, J., Panter, K.E., Patel, J., Smith, D.M., Spangenberger, A., Freeman, B. 2008. The Fetal Cleft palate: V. Elucidation of the Mechanism of Palatal Clefting in the Congenital Caprine Model. Plastic and Reconstructive Surgery, pp.1328-1334 Pfister, J.A., Lee, S.T., Panter, K.E., Motteram, E.S., Gay, C. 2008. Effects of Experience and Lactation on Lupine Consumption by Cattle. Rangeland Ecology and Management 61:240-244 March 2008 Hanes, M.C., Weinzweig, J., Panter, K.E., Mcclellan, T., Caterson, S.A., Buchman, S.R., Faulkner, J.A., Yu, D., Cederba, P.S., Larkin, L.M. 2008. The Effect of Cleft Palate Repair on Contractile Properties of Single Permeabilized Muscle Fibers From Congenitally Cleft Goats Palates. Annals of Plastic Surgery, Vol. 60 No. 2, pp. 188-193. Hanes, M.C., Weinzweig, J., Kuzon, W.M., Panter, K.E., Buchman, S.R., Faulkner, J.A., Yu, D., Cederna, P.S., Larkin, L.M. 2007. Contractile properties of single permeabilized muscle fibers from congenital cleft palates and normal palates of Spanish goats. Plastic and Reconstructive Surgery, 119:1685 - 1694. Rader, E.P., Cederna, P.S., Weinzweig, J., Panter, K.E., Yu, D., Buchman, S.R., Larkin, L.M., Faulkner, J.A. 2007. Contraction-induced injury to single permeabilized muscle fibers from normal and congenitally-clefted goat palates. Cleft Palate-Craniofacial Journal, Vol. 44 No. 2 pp. 216-222. Gorniak, S.L., Pfister, J.A., Lanzoniz, E.C., Raspantini, E.R. 2008. A note on averting goats to a toxic but palatable plant, Leucaena leucocephala. Applied Animal Behaviour Science, Vol. 111, No. 3-4, pp. 306-401. Ralphs, M.H., Panter, K.E., Gay, C., Motteram, E., Lee, S.T. 2007. Cattle grazeing velvet lupine (lupinus leucophyllus): influence of associated forages, alkaloid levels and population cycles. Poisonous Plant Global Research and Solutions, Chpt. 67, pp. 401-406 Thacker, E., Ralphs, M.H., Call, C., Benson, B., Green, S. 2008. Invasion of Broom Snakeweed (Gutierrezia sarothrae) Following Disturbance: Evaluating Change in a State-and-Transition Model. Rangeland Ecology and Management, Vol. 61, Issue 3, pp. 263-268. DOI:10:2111/07.043.1 Ralphs, M.H., Wiedmeier, R.D., Banks, J.E. 2007. Decreasing forage allowance can force cattle to graze broom snakeweed (gutierrezia sarothrae) as a potential biological control. Rangeland Ecology and Management, 60:487-497.