Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Grazing of ponderosa pine needles, broom snakeweed and certain juniper species by pregnant cows may result in abortion/premature parturition, retained placentas and endometritis which causes substantial economic losses to cattle producers in the western U.S. and Canada. Lodgepole pine and Monterey Cypress also cause abortion in cattle. Recent significant losses have been reported during the 2001-2004 seasons. While pine needles and junipers predominantly affect cattle, broom snakeweeds also cause toxicosis and abortion in sheep and goats in the southwest. Research on these problems is being addressed using a multidisciplinary approach requiring research on five main objectives: 1. Isolate and characterize the abortifacient and toxic components of pine needles and broom snakeweed. Determine the
metabolism and clearance times of the abortifacient and toxic compounds and describe their physiologic activity. 2. Describe pathophysiologic, endocrine, immunologic, and morphologic changes associated with the abortion; determine effects on uterine function; define mechanisms and determine effects on neonatal survival and postpartum interval; and develop treatment for abortion sequelae. 3. Determine animal, environmental, and management factors influencing consumption and develop strategies to reduce losses. Define the conditions which cause livestock to eat ponderosa pine needles and broom snakeweed and develop management strategies to prevent poisoning. 4. Identify toxins, describe their structure activity relationships, characterize toxicokinetics, and delineate the mechanisms of action of plant teratogens. 5. Describe environmental, nutritional or management conditions which cause livestock to graze teratogenic plants during susceptible periods of gestation. Define
management strategies that will reduce losses. Pine needle abortion from Ponderosa pine (PN) results in an estimated loss of over $20 million annually to cattle producers in the western U.S. Pine needles cause abortions/premature births and retained placenta in cattle when ingested during the last trimester of pregnancy. Also, ponderosa pines are rapidly invading grasslands on forest boundaries reducing grass production and inhibiting maximum efficiency of many of these ranges. The problem is pervasive in the western United States and Canada where cattle graze in pine forests. Large losses have been reported in South Dakota, Wyoming, Montana, Oregon, California, Idaho, New Mexico, Arizona etc. Lodgepole pine has recently been shown to cause abortion in cattle and losses have been reported in western Canada. In the spring of 2003, large losses in California were attributed to California Juniper (an evergreen shrub containing high concentrations of isocupressic acid, the abortifacient
compound). These losses had occurred for years but only in the spring of 2003 were the losses linked to juniper. Undoubtedly, other pines that contain the abortifacient toxin isocupressic acid (ICA) contribute to losses on a regional, national and international basis. In Australia and New Zealand, the tree Cupressus macrocarpa (Monterey Cypress), causes similar abortions in cattle and in Korea, Pinus koraiensis has cause abortion in cattle. Recent reports of abortions in dairy cows in the eastern U.S. are believed to be linked to ingestion of Monterey Cypress ingestion. Chemical analysis of these species and others at the Poisonous Plant Research Lab has confirmed ICA as the primary abortificient in pine needles. ICA was identified as the primary compound but it is rapidly metabolized in the cow and disappears after the first pass through the liver. Therefore, the metabolite acting at the target tissue and mechanism of action are unknown. Direct losses associated with the abortion do
not take into account other losses such as forage, management, costs of purchasing replacements, cows deaths, added veterinary care etc. In the southwestern U.S. broom snakeweed (Gutierrezia spp.) causes annual estimated losses of over $40 million to the livestock industry. These losses include toxicoses, abortion, retained placenta and lost production in cattle, sheep and goats and loss of forage production from snakeweed invasion. In addition, because of its allelopathic nature, broom snakeweed aggressively invades grasslands, decreasing available good forage for livestock, compromising native plant biodiversity and inhibiting optimum utilization of many of these rangelands. Lupinus spp. continue to cause significant losses to the livestock industry in the western U.S. from induced crooked calf syndrome. Lupine research at the Poisonous Plant Research Lab began in about 1958 and subsequent research identified the cause, determined the time of insult, identified the toxins, and
developed a management strategy. However, considerable losses continue to occur and in the spring of 2003, large losses were reported in California, Oregon, Idaho, western Canada and Washington State. We have done extensive research over the last 3 years in the Scabland region of eastern Washington where lupines are widespread and have caused catastrophic losses in recent years. Grazing studies in eastern Washington and collaborative feeding trials at Washington State University and the Poisonous Plant Research Lab have provided new and important information to help cattle producers better manage grazing programs to reduce losses. It is important that grazing studies continue to observe altered patterns of lupine growth and animal grazing behavior with changing environmental conditions. While lupines cause large losses, other teratogenic plants such as poison-hemlock, Nicotiana spp. etc. also contribute to the overall losses from poisonous plants to the livestock community.
Teratogenic plants, especially lupines cause large losses annually to the livestock industry in the western US and western Canada. Catastrophic losses from lupines have occurred recently in certain regions of Oregon, the Channel Scablands of eastern Washington State and southeastern Idaho. In Central Oregon, over 60% of the calf crop in one herd was lost, in the spring of 2003 in southeastern Idaho 55% of the calves born had to be destroyed and in 1997 over 4,000 calves in Adams County, Washington were destroyed; because of birth defects resulting in a $1.7 million direct economic loss and $5 million indirect loss to Adams county alone. Smaller, individual losses have been frequently reported over the last 5 years from most states of the western U.S. and Canada. These losses do not take into account many of the increased management costs associated with such large losses, for example increased death losses, increased culling practices, heifer replacement costs, lost grazing
opportunities, fencing, additional feed, and the emotional stress. Through chemical analysis the Poisonous Plant Research Lab has identified many individual toxins and metabolites in lupines, pinus species, juniper species, broom snakeweed etc. Physiologic/toxic activity has been determined in target species and rodent models. An ELISA has been developed for some of the toxins and certain metabolites found in pine needles and juniper. Some of these toxins have been screened in vitro for biological activity using oocyte/embryo cultures. Using ELISA and sophisticated chemistry technology, the toxin, isocupressic acid (ICA, the abortifacient toxin in pines/junipers), and selected metabolites of ICA were identified in blood serum of pregnant cows. Absorption and disappearance of ICA and its metabolites will be determined in blood. An ELISA was developed for the teratogenic steroidal alkaloids cyclopamine and jervine in blood and animal tissues. Teratogenic and toxic alkaloids have been
identified in lupines and other plants and absorption and elimination profiles in animal serum are being investigated. This research will provide information and guidelines to improve the quality of feeds for livestock, enhance product quality for consumers of animal products and better prognosis of disease. Management strategies and diagnostic tools (ELISAs) are being developed to identify toxicoses from pine needles, lupines and other teratogenic plants. This research will provide information to assist in the prevention of plant induced birth defects. Over the last 3 years grazing studies have been done to determine why and when cattle graze pine needles and lupines. Forage quality, environmental factors and animal grazing behavior have been monitored to determine conditions when cattle graze these plants. This research will enhance grazing strategies to prevent birth defects from recurring and prevent the associated economic losses to the livestock industry and enhance the economic
base of ranchers and the communities in which they live. 2. List the milestones (indicators of progress) from your Project Plan. This project CRIS was initiated in FY 2000 as a 5 year project; therefore, the milestones in the project plan encompass 5 years. Objective 1 Milestone 1.1 Synthesize gathic acid and dehydroagathic acid (first pass ICA metabolites) to test for abortifacient activity in cows, 2001-2002; dose pregnant cows with the synthesized metabolites, 2002-2004. Milestone 1.2 - Develop analytical methods for analysis of diterpene acids in Broom Snakeweed (BSW) and collect BSW from various locations and determine their respective diterpene acid profiles, 2002-2004; Extracts will be fed by gavage to pregnant cows to determine abortifacient activity, 2002-2005. Objective 2 Milestone 2.1 - Cows will be treated with various drugs such as the prostaglandin inhibitors, blood flow regulators and reproductive hormones to help determine mechanism of action of toxins, 2000-2001;
Blood samples will be analyzed for progesterone, estradiol, etc. 2002-2004; Data will be analyzed and published, 2003-2005. Milestone 2.2 - Cows will be bred and serial vaccinations done using isocupressic acid conjugates through pregnancy. Blood samples will be drawn and serum titers determined, 2001-2002; Vaccinated cows will be challenged with pine needles and blood samples drawn for hormone analyses, 2003-2005. Milestone 2.3 - Cows with different body condition scores will be fed fresh broom snakeweed 2003-2005. Milestone 2.4 - Cows will be treated with various therapies after abortions, 2000-2004. Objective 3 Milestone 3.1 - Survey ponderosa pine populations for abortifacient compounds and determine their change over time, by geographical site, and from weather conditions, 2001-2002. Milestone 3.2 - Impact of energy and protein status on susceptibility to pine needle (PN) abortion, 2000-2001. Milestone 3.3 - Cows will be averted to green and dry PN, 2002-2003. Milestone 3.4 -
Influence of snow cover, temperature, forage height and standing crop on consumption of PN, 2003-2004. Objective 4 Milestone 4.1 - Determine the relative toxicity and teratogenicity of the nine principle piperidine alkaloids (from Conium, Lupinus, and Nicotiana) in mice and goats, respectively, 2001-2004; Goats will be bred and specific alkaloids from Conium, Lupinus, and Nicotiana will be tested for teratogenic activity, 2003-2005. Objective 5 Milestone 5:1 - Lupine samples will be collected in Washington State and other locations where crooked calf disease occurs and submitted for analysis, 2000-2005; Risk assessment will be established using alkaloid profiles and concentrations, 2003-2005; Management strategies will be correlated with risk assessments, 2005. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Objective 1:1.1 The isocupressic acid (ICA)
metabolites from pine needles (agathic acid and dehydroagathic acid) have been isolated and synthesized and screened for cytotoxicity using bovine embryos. Milestone Substantially Met 2. Objective 1:1.2 Crude extracts from broom snakeweed have been analyzed and compounds similar to the abortifacient compounds in pine needles have been identified. Milestone Substantially Met 3. Objective 2:2.1 Pregnant cows were treated with seven drug regimens to determine if the mechanism of action of pine needle abortion could be blocked. Milestone Fully Met 4. Objective 2:2.2 An isocupressic acid conjugate and subsequent vaccine was developed and administered to pregnant cows to reduce pine needle abortions. Milestone Fully Met 5. Objective 2:2.3 Two groups of cows were preconditioned, one in low body condition and one in a high body condition to compare acceptance of pine needles or broom snakeweed. Milestone Fully Met 6. Objective 2:2.4 Four different therapies were tested in cows following pine
needle- induced abortion to determine the optimum treatment for retained fetal membranes to enhance post- partum breeding. Milestone Substantially Met 7. Objective 3: 3.4 Grazing behavior of pregnant cows on ranges heavily infested with ponderosa pine was observed. Milestone Fully Met 8. Objective 4: 4.1 Individual alkaloids with suspected teratogenic activity from lupines (ammodendrine), poison-hemlock (coniine) and Nicotiana spp. (anabasine) were toxicity tested in a mouse bioassay. Milestone Substantially Met 9. Objective 5: 5.1 Analysis of lupine samples collected from the Scabland Region of eastern Washington State during grazing seasons 2000 - 2003 have been completed. Alkaloid profiles and risk assessment for potential toxicoses have also been completed. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Objective 1: FY
2005 - FY 2007; Further identify and scale-up the synthesis of isocupressic acid metabolites. Broom snakeweed extracts from fresh plants will be analyzed and pregnant cows will be treated with these extracts. Abortifacient action and hormone profiles will be determined in these cows. Because of the scope of this project and the use of pregnant cows as the only acceptable model, this work will need to continue through FY 2006 and FY 2007. Objective 2: FY 2005; Blood serum titers will be evaluated from cows vaccinated with ICA conjugates. Evaluation of data on preferred therapies for retained fetal membranes as a result of pine needle-induced abortion will be completed. FY 2006-FY 2007; Because of the scope and logistics of using pregnant cows as the animal model, broom snakeweed feeding trials in thin vs fat cows will not occur until FY 2006. Objective 3: Milestones listed in objective 3 have been accomplished, however, further research to determine what environmental and/or animal
factors contribute to the ingestion of pine needles or broom snakeweed will continue in FY 2005 - FY 2007. Objective 4: FY 2005; Chemical isolation, purification and characterization of nine piperidine alkaloids and certain enantiomers of selected alkaloids will continue. Toxicity testing will continue using a mouse bioassay to compare individual alkaloids and their enantiomers. FY 2006 - FY 2007; Selected piperidine alkaloids and their enantiomers will be tested in the goat and the kinetics evaluated in the fetal compartment. Objective 5: FY 2005; Lupine samples will be collected from predetermined sites in the scabland region of eastern Washington State where lupine induced crooked calf disease continues to cause large losses to the livestock industry of that region. Selected lupines samples will be collected in other key areas for comparison. Grazing studies to determine why and when cattle graze lupines will continue. FY 2006- FY 2007; Data collected over the last 3 years will
be analyzed and evaluated to develop management strategies to reduce losses in eastern Washington. Year 1: 1) Continue to conduct grazing studies in the Scabland Region of eastern Washington and the mountain meadows of southern Idaho to determine what factors (plant, environment or animal) affect cattles propensity to graze lupine; 2) Prepare a draft summary bulletin with current information and recommendations for grazing management of lupine on the Scablands of eastern Washington; 3) Individual quinolizidine and piperidine alkaloids will be isolated from lupines and other teratogenic plants for comparative toxicity/teratogenicity testing in a mouse bioassay; 4) Continue to monitor lupine populations in the Scabland Region of eastern Washington to evaluate the effects of changing environmental factors on lupine density; 5) Continue to explore scale-up synthesis of ICA metabolites from pine needles for cattle feeding trials; 6) Continue investigating the toxic and abortifacient
activity of broom snakeweed; 7) Continue ecology study to determine long-term impact of fire and grazing on snakeweed populations. 8) Profile the diterpene acids present in broom snakeweed plants. Year 2: 1) Continue the chemical investigation of broom snakeweed for abortifacient and toxic compounds; 2) Evaluate serum from cattle after broom snakeweed consumption using the recently developed enzyme-linked immuno assay (ELISA) procedure to identify possible structurally related compounds that are abortifacient; 3) Continue investigation of immunologic activity of ICA in an effort to develop a vaccine in cows; 4) Continue collaborations with Washington State to further study the lupine- induced crooked calf disease problem on the vast rangelands of the Scabland Region. Year 3. 1) Continue screening broom snakeweed plants for abortifacient compounds; 2) Continue investigation of abortifacient metabolites in the serum of cattle fed pine needles or common juniper; 3) Scale up synthesis
of the same compounds for animal feeding trials. 4) Grazing studies will continue on the Channel Scablands of eastern Washington to determine when and why cattle graze lupines; 5) Management strategies will be developed and recommendations will be given to ranchers in the Scabland Region to reduce calf losses. 4a What was the single most significant accomplishment this past year? Lupine Alkaloid Research: Individual alkaloids with suspected Teratogenic activity from Lupines (ammodendrine), poison hemlock (coniine) and Nicotiana spp. (anabasine) were tested in a mice bioassay, + and enantiomers of ammodendrine were isolated and optical rotations of the enantiomers defined. Structural characteristics of each of these alkaloids is very important in predicting risk of poisonous plant-induced birth defects in livestock. Furthermore, the optical rotation of a pair of enantiomeres appears to be important on risk analysis of some of the plants containing these compounds. A publication
reporting these findings has been published and a second publication has been submitted. 4b List other significant accomplishments, if any. Cattle Grazing Lupine Research: Twelve lactating and non-lactating cows (n=6) were grazed together for 19 days on a 2.4 ha pasture divided into 2 paddocks. This pasture was a very productive swale with abundant forage (> 2000 kg/ha). Lupine density varied from 1-2 plants/m2. Cattle began eating lupine on day 4 and consumption generally increased until day 9 when lupine availability was depleted. There was no difference (p> 0.6) in consumption between lactating and dry cows. Lupine consumption peaked at 10-15% of the diets. Cattle were then switched to the next paddock and grazed for 9 days until lupine was depleted. Cattle ate mostly grasses on the first day after switching paddocks, then lupine consumption averaged 5-15% of diets during the remainder of the trial. Other forbs comprised the majority (60- 70%) of the diets, and consumption of dry
grass varied from ~10-20% of the diets. These same cows were then moved to SE Idaho on lupine infested rangelands for a 2 week trial, but they ate essentially no lupine during that period. Biological Mechanism of Veratrum Alkaloids: Cyclopamine is a toxic principal in Veratrum californicum and the cause of cyclopic lamb malformations in sheep. This compound is of great interest because of its biochemical mode of action and its ability to block sonic hedge hog protein signaling pathways which now appear to be active in certain cancers. Its potential therapeutic use is of great interest. Cyclopamine is not readily available commercially and historically this laboratory has provided material to the research community for experimental purposes. A large collection of root material was collected last fall to supply additional source material. Secondly, analytical procedures were developed for analysis of cyclopamine in serum samples. Analysis of serum levels will be useful in determining
clearance times and tissue levels in experimental and therapeutic treatments of malignant tumors. 4d Progress report. Lupinus spp. continue to cause significant losses to the livestock industry in the western U.S. from induced crooked calf syndrome. Lupine research at the Poisonous Plant Research Lab began in about 1958 and subsequent research identified the cause, determined the time of insult, identified the toxins, and developed a management strategy. However, considerable losses continue to occur and in the spring of 2003, large losses were reported in California, Oregon, Idaho, western Canada and Washington State. We have done extensive research over the last 3 years in the Scabland region of eastern Washington where lupines are widespread and have caused catastrophic losses in recent years. Grazing studies in eastern Washington and collaborative feeding trials at Washington State University and the Poisonous Plant Research Lab have provided new and important information to help
cattle producers better manage grazing programs to reduce losses. It is important that grazing studies continue to observe altered patterns of lupine growth and animal grazing behavior with changing environmental conditions. While lupines cause large losses, other teratogenic plants such as poison-hemlock, Nicotiana spp. etc. also contribute to the overall losses from poisonous plants to the livestock community. Grazing of ponderosa pine needles, broom snakeweed and certain juniper species by pregnant cows may result in abortion/premature parturition, retained placentas and endometritis which causes substantial economic losses to cattle producers in the western U.S. and Canada. Lodgepole pine and Monterey Cypress also cause abortion in cattle. Recent significant losses have been reported during the 2001-2004 seasons. While pine needles and junipers predominantly affect cattle, broom snakeweeds also cause toxicosis and abortion in sheep and goats in the southwest. Research on these
problems is being addressed using a multidisciplinary approach requiring research on five main objectives: 1. Isolate and characterize the abortifacient and toxic components of pine needles and broom snakeweed. Determine the metabolism and clearance times of the abortifacient and toxic compounds and describe their physiologic activity. 2. Describe pathophysiologic, endocrine, immunologic, and morphologic changes associated with the abortion; determine effects on uterine function; define mechanisms and determine effects on neonatal survival and postpartum interval; and develop treatment for abortion sequelae. 3. Determine animal, environmental, and management factors influencing consumption and develop strategies to reduce losses. Define the conditions which cause livestock to eat ponderosa pine needles and broom snakeweed and develop management strategies to prevent poisoning. 4. Identify toxins, describe their structure activity relationships, characterize toxicokinetics, and
delineate the mechanisms of action of plant teratogens. 5. Describe environmental, nutritional or management conditions which cause livestock to graze teratogenic plants during susceptible periods of gestation. Define management strategies that will reduce losses. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Components of this research are found under National Programs 205 Rangeland, Pasture, and Forages and directly contributes to research Component V Integrated Management of Weeds and Other Pests, the Poisonous Plant area, and 103 Animal Health Component IV Toxic Plants. Research activities are further related to ARS Strategic Plan Goal 3.1.3.2 Animal Offspring Reared, Develop technology to improve reproductive efficiency and increase the number of offspring (and eggs) reared per female animal per year, and ARS Strategic Plan Goal 3.2.3.2 Natural and Synthetic Chemicals - Animals, Develop knowledge about ways of
controlling the toxic effects of natural and synthetic chemicals in animals to reduce lossses and illegal product residues. * Research at the Poisonous Plant Research Lab on Veratrum californicum and lupines began in the early 1950's; these plants were identified as the cause of the malformed lamb syndrome and crooked calf disease respectively. * Toxins and teratogens from Veratrum and lupines were identified and characterized. * ELISAs have been developed for detection of ICA and its sera metabolites and cyclopamine and jervine (teratogens from Veratrum) using polyclonal antibodies. Using these tools and simple extraction techniques, rapid screening of biological samples for the presence of these toxins and/or their metabolites was accomplished. * A collaborative study with the University of Idaho on absorption and elimination of lupine alkaloids in sheep differing in body condition showed that thin sheep more rapidly absorbed toxic alkaloids than did fat sheep, and conversely thin
sheep showed slower elimination of alkaloids compared to fat animals. * Grazing trials with fat and thin cattle indicated that thin cattle initially ate more lupine than did fat animals in eastern Washington. Eventually all animals ate substantial quantities of lupine, but thin animals ate more lupine at times, thus the risk of intoxication is particularly high for thin animals compared to cattle in higher body condition. *Isolated and identified the abortifacient toxin in pine needles as a labdane resin acid, isocupressic acid (ICA). Major serum metabolites were identified as dihydroagathic acid, agathic acid, imbricatoloic acid, and tetrahydroagathic acid. Maximum serum metabolites occured between 24 and 48 hours after consumption of pine needles. * Isocupressic acid was identified in Cupressus macrocarpa (Monterey Cypress), Pinus contorta (lodgepole pine), Pinus koriensis, (Korean Pine) and Juniperus communis (common juniper). In subsequent feeding trials lodgepole pine and
common juniper were demonstrated to be abortifacient. * Over 25 tree and shrub species from throughout the western and southern states have been analyzed for ICA. Significant levels (>0.5% dry weight) were detected in Pinus ponderosa, P. jefferyi, P. contorta (lodgepole pine), Juniperus scopulorum (Rocky Mountain juniper) J. communis (common juniper) and J. californicum (California juniper). Similar toxins have been identified in broom snakeweed but the putative abortifacient compound has not yet been identified. Identification of ICA, its metabolites and quantitative analysis in the pine needles provides useful information for the range scientists and ranchers in formulating management practices to reduce or prevent losses. * Grazing and pen studies examined conditioned food aversions as a potential tool to keep cattle from consuming pine needles. Cattle were successfully averted to green needles initially, but the aversion extinguished as cattle began consuming dry needles and
then reverted back to the green needles. In nutrition trials, low energy status initially influenced cows to eat more needles but as cows gained experience with needles this treatment effect diminished; supplemental protein did not result in decreased pine needle consumption. *Using in vitro fertilization techniques, oocytes and embryos were cultured in the presence of isocupressic acid (the pine needle toxin) or various steroidal alkaloid teratogens including cyclopamine from Veratrum californicum, a synthetic cyclopamine derivative and three steroidal alkaloid teratogens from Solanum. Steroidal alkaloid teratogens from Veratrum and Solanum spp. inhibited embryo growth and development in vitro when introduced in the culture media at different concentrations. Isocupressic acid enhanced oocyte maturation and early embryo growth and development. ICA-treated embryos were transferred to recipient cows, and while pregnancy progressed in a normal fashion, 2 of 5 calves were born dead and
necropsy revealed an identical cardiovascular anomaly in both calves. While the increase in embryo development induced by ICA may have significant utility in IVF systems, further evaluation is required. *The third year of a 5 year grazing study was conducted in eastern Washington to determine when cows eat lupine (Lupinus leucophyllus) and correlate consumption to the critical period of gestation which causes the crooked calf syndrome. In all 3 years, cows started eating lupine around the first of July after cheatgrass had matured, and the other forbs were grazed out or matured lupine consumption ranged from 17 to 25% of diets). This period of consumption overlaps the critical period of gestation (40-70 day of pregnancy) if the traditional breeding season begins May 1, and cattle should be removed from lupine areas during this period. *Cows that were positively conditioned to graze snakeweed were kept from the previous year's study to determine if limiting the amount of feed could
force them to graze snakeweed. The cows were fenced into small lanes with only enough herbaceous forage to supply 25% of their daily intake requirement and were moved to a new lane each day. Cows grazed out the green grass first, then dry cheatgrass, then grazed snakeweed. Almost all snakeweed plants were grazed and 45% of its biomass was removed. *Broom snakeweed is an invasive native plant that inhibits growth of more desirable species and may cause abortions in cows, sheep and goats. Heifers were positively conditioned to preferentially graze snakeweed as a biological tool to suppress it in the sagebrush steppe plant community in northern Utah. The heifers were fed snakeweed then given starch as a positive energy feedback to enhance their acceptance of snakeweed. Conditioning was successful in the pen as starch-fed heifers consumed more snakeweed than untreated controls, but the heifers would not graze snakeweed in the field until all other forage was consumed. Then the positively
conditioned heifers consumed more snakeweed than the controls. *Cattle in high (7.5 on 9 point scale) and low (3.5) body condition were fed green pine needles free choice during a 2-week period. Cattle in low (i.e., thin) body condition ate more (P=0.001) pine needles than did fat cows (5.5 g pine needles/kg body weigt vs. 1.0 g/kg, respectively). *Cows that were positively conditioned to graze snakeweed were kept from the previous year's study to determine if limiting the amount of feed could force them to graze snakeweed. The cows were fenced into small lanes with only enough herbaceous forage to supply 25% of their daily intake requirement and were moved to a new lane each day. Cows grazed out the green grass first, then dry cheatgrass, then grazed snakeweed. Almost all snakeweed plants were grazed and 45% of its biomass was removed. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-
user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Management to reduce livestock losses from poisonous plants and Influence of drought on losses to toxic plants were presented at the Arizona Strip Workshops, March 26, 2003, Kanab Utah, and March 27, 2003, St. George, Utah. Sponsored by Arizona and Utah Extension Services. "Local Poisonous Range Plants" was presented to the Shoshone-Bannock tribes at the Northwest Intertribal Agriculture Council Conference, Pocatello, Idaho, March 16-18, 2005. The information presented at these workshops is available to ranchers and is being implemented in many grazing programs. There is considerable interest from scientists doing research on humans in using cyclopamine (the teratogen from Veratrum californicum) and its ability to block the effects of a certain family of genes known as sonic hedgehog. Purified crystalline cyclopamine has been prepared from V.
californicum and shared with nine different research organizations to be used in a number of different in vitro studies designed to investigate the role of the hedgehog pathway. Examples include the study of specific genes in the sonic hedgehog pathway in the cerebellum; effects of pesticides on crustacean development and shell formation; mechanism of action of the anti-tumor drug tamoxifen; the regulation of neural tissue repair; studies on craniofacial neural crest cells; and hedgehog pathway activity in medulloblastoma growth. Information has been transferred to other scientists through publications in peer-reviewed journals and to scientists, land managers, extension agents and ranchers through popular publications, seminars, workshops, internet, e-mail and telephone conversations. This information is now available for ranchers use to develop grazing strategies to reduce losses. As new information is made available, producer meetings are held to transfer new information and
technologies. The major constraints to adoption of current technology include changing environmental conditions, forage availability and acceptance of new management programs. Results of lupine research have frequently been discussed with extension agents and ranchers in many western states. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Panter, K.E., James, L.F., Wierenga, T., Gay, C., Motteram, E., Lee, S.T., Gardner, D.R., Pfister, J.A., Ralphs, M.H. and Stegelmeier, B.L. 2005. Lupine research at the poisonous plant research laboratory: Past, present and future. 7th International symposium on Poisonous Plants. Logan, UT. June 6-10. Pfister, J.A. and Panter, K.E. attended the 1st Latin American Symposium on Poisonous Plants and Dr. Panter presented "Poisonous Plants: Effects on Reproduction and Embryo growth and Development". Panter, K.E.
and Gardner, D.R. presented "Selected Poisonous Plants at the Northwest Intertribal Agriculture Council Conference of the Shonshonee/Bannock Tribe, March 16-18, 2005. Lee, S.T., Panter, K.E., Molyneux, R.J., Chang, C.W.T., Gardner, D.R. and Pfister, J.A. 2005. Separation and isolation of teratogenic piperidine enantiomers from Lupinus and Nicotiana species. 7th Internation Symposium on Poisonous Plants. Logan, Utah. June 6-10. Ralphs, M.H., Panter, K.E., ay, C.C., Motteram, E.S., and Lee, S.T. 2005. Cattle consumption of velvet lupine in the channel scablands of eastern Washington. 7th International Symposium on Poisonous Plants. Logan, Utah. June 6-10. Pfister, J.A., Panter, K.E., Lee, S.T., and Gay, C.C. 2005. Effects of lactation and experience on cattle grazing of lupine. 7th International Symposium on Poisonous Plants. Logan, Utah. June 6-10. Gay, C.C., Panter, K.E., Motteram, E.S., Gay, J.M., Wierenga, T., and Platt, T. 2005. Risk factors for lupine-induced
arthrogryposis (crooked calf disorder). 7th International Symposium on Poisonous Plants. Logan, Utah. June 6-10. Gay, C.C., Panter, K.E., Mealey, K.L., Gay, J.M., Hfartarson, S.W., Tibary, A., Motteram, E.S., Wierenga, T., and James, L.F. 2005. Effect of repeated challenge of the plasma disposition of alkaloids in cattle that have given birth to calves with lupine-induced arthrogryposis or to clinically normal calves. 7th International Symposium on Poisonous Plants. Logan, UT. June 6-10. Barkworth, M.E.,, Peip, M., Fogel, R., Lim, S.J., Pfister, J.A., and Panter, K.E. 2005. Poisonous Plants of the West: Developing a multifaceted web resource. 7th International Symposium on Poisonous Plants. Logan, Utah. June 6-10 Gunn, D. and Panter, K.E. 2005. Poisonous range plants on the Fort Hall Reservation in southeastern Idaho. 7th International Symposium on Poisonous Plants. Logan, Utah. June 6-10. Motteram, E.S., Gay, C.C., Panter, K.E., and Platt, T. 2005. A geographic
look at the 1997 crooked calf outbreak in Adams County, Washington using geographic information systems *GIS) technology. 7th International Symposium on Poisonous Plants. Logan, Utah. June 6-10. Motteram, E.S., Gay, C.C., Panter, K.E., and Wierenga, T. 2005. Year to year variation in total alkaloid and anagyrine concentration in Lupinus leucophyllus growing on the scablands of central Washington. 7th International symposium on Poisonous Plants. Logan, Utah. June 6-10. Launchbaugh, K. Pfister, J.A., LOpez, S., and Frost, R. 2005. Body condition affects blood alkaloid and monoterpene kinetics and voluntary intake of chemically-defended plants by livestock. 7th International Symposium on Poisonous Plants. Logan, Utah. June 6-10. Gay, C.C., Panter, K.E., Motteram, E.S., Gay, J.M., Wierenga, T. and Platt, T. 2005. Effect of shade on alkaloid content of Lupinus leucophyllus. 7th International Symposium on Poisonous Plants. Logan, Utah. June 6-10.
Impacts
(N/A)
Publications
- James, L.F., Panter, K.E., Gaffield Jr, W.P., Molyneux, R.J. 2004. Biomedical applications of poisonous plant research. Journal of Agricultural and Food Chemistry.
- Lee, S.T., Molyneux, R.J., Panter, K.E., Chang, C., Gardner, D.R., Pfister, J.A., Garrossian, M. 2005. Structure-activity relationships of ammodendrine and n-methyl ammodendrine enantiomers: isolation, optical rotation, and toxicity. Journal of Natural Products.
- Panter, K.E., Wang, S., Gaffield Jr, W.P., James, L.F., Evans, R.C., Bunch, T. 2005. Effects of cyclopamine, a veratrum alkaloid, and its synthetic analog cyclopamine-4-ene-3-one on in vitro bovine oocyte maturation and subsequent embryo development. Western Section of Animal Science Proceedings.
- Karhadkar, S.S., Bova, G.S., Abdallah, N., Dhara, S., Gardner, D.R., Maltra, A., Isaacs, J.T., Berman, D.M., Beachy, P.A. 2004. Hedgehog signalling in prostate regeneration, neoplasia and metastasis. Nature.
- KNUBEL, B.F., PANTER, K.E., PROVENZA, F.D. PREGNANCY IN GOATS DOES NOT INFLUENCE INTAKE OF NOVEL OR FAMILIAR FOODS WITH OR WITHOUT TOXINS. APPLIED ANIMAL BEHAVIOR SCIENCE. 2004.
- Wang, S., Panter, K.E., Gaffield, W., Bunch, T. 2004. Effects of steroidal glycoalkaloids from potatoes (solanum tuberosum) on in vitro bovine embryo development. Animal Reproduction Sciences.
- Wang, S., Panter, K.E., Gardner, D.R., Evans, R.C., Bunch, T.D. 2004. Effects of the pine needle abortifacient, isocupressic acid, on bovine oocyte maturation and preimplantation embryo development. Animal Reproduction Sciences.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Lupine research at the Poisonous Plant Research Lab began seriously in about 1958. Lupines continue to cause significant losses to the livestock industry in the western U.S. from induced 'crooked calf syndrome'. In the spring of 2003, large losses were reported in California, Oregon, Idaho, Western Canada and Washington State. We have done extensive research over the last 3 years in the Scabland region of Eastern Washington where lupines are widespread and cause large losses every year to cattle producers. Recent grazing studies in eastern Washington and collaborative feeding trials at Washington State University and the Poisonous Plant Research Lab have provided new and important information to help cattle producers better manage grazing programs to reduce losses. It is important that grazing studies continue to observe changing patterns of lupine growth and animal grazing behavior
as environmental conditions change. While lupines cause large losses, other teratogenic plants including poison-hemlock, Nicotiana spp. etc. also contribute to the overall losses from poisonous plants to the livestock community. Grazing of ponderosa pine needles, broom snakeweed and certain junipers by pregnant cows results in abortion/premature parturition, retained placentas and endometritis causing substantial economic losses to cattle producers in the western U.S. and Canada. Lodgepole pine and Monterey Cypress also cause abortion in cattle. Recent significant losses have been reported during the 2001-2003 seasons. While pine needles and junipers apparently predominantly affect cattle, broom snakeweeds also cause toxicosis and abortion in sheep and goats in the south west. Research on these problems is being addressed using a multidisciplinary approach requiring research on five main objectives: ' Describe physiologic activity in animals and determine serum profiles of
absorption, metabolism and clearance times of abortifacient and teratogenic compounds and potentially active metabolites from animal tissues. ' Isolation and characterization of abortifacient, toxic and teratogenic compounds in pine needles, broom snakeweed, lupine and other teratogenic plants, identifying metabolic or detoxification derivatives found in the animal that exhibit abortifacient or teratogenic activity. ' Determine animal, environmental, and management factors influencing consumption of ponderosa pine needles, broom snakeweed and lupines and develop strategies to reduce losses. ' Develop conjugates or immunogenic derivatives to the abortifacient and teratogenic compounds and evaluate potential vaccines, antitoxins and toxin scavengers and determine what treatments can be applied to reduce losses from premature or deformed offspring and retained placentas. ' Define the mechanism of action or how pine needles/broom snakeweed cause the abortions and how teratogenic
plants cause birth defects. Describe pathophysiology, endocrine, immunologic and morphologic changes associated with these conditions. 2. How serious is the problem? Why does it matter? Teratogenic plants and especially lupines cause large losses annually to the livestock industry in the western US and western Canada. Catastrophic losses from lupines have occurred recently in certain regions of Oregon, the Channel Scablands of eastern Washington State and south eastern Idaho. In Central Oregon, over 60% of the calf crop in one herd was lost, in the Spring of 2003 in south eastern Idaho 55% of the calves born to a set of heifers syncronized in estrus and bred to AI bulls had to be destroyed and in 1997 over 4,000 calves in Adams County Washington were destroyed because of birth defects. These losses do not take into account many of the increased management costs associated with such large losses, for example increased death losses, increased culling practices, heifer replacement
costs, lost grazing opportunities, fencing, additional feed, and the emotional loss. Pine needle abortion from Ponderosa pine (PN) cause an estimated loss of over $20 million annually to cattle producers in the western U.S. Pine needles cause abortions/premature births and retained placenta in cattle when ingested during the last trimester of pregnancy. Also, ponderosa pines are rapidly invading grass lands on forest boundaries reducing grass production and inhibiting maximum efficiency of many of these ranges. The problem is pervasive in the western United States and Canada where cattle graze in pine forests. Large losses have been reported in South Dakota, Wyoming, Montana, Oregon, California, Idaho, New Mexico, Arizona etc. Lodgepole pine has recently been shown to cause abortion in cattle and losses have been reported in western Canada. In the spring of 2003, large losses in California were attributed to California Juniper ( an evergreen shrub containing high concentration
of isocupressic acid). These losses had occurred for years but only the spring of 2003 were the losses linked to juniper. Undoubtedly, other pines that contain the abortifacient toxin isocupressic acid (ICA) contribute to losses on a regional, national and international basis. In Australia and New Zealand the tree Cupressus macrocarpa causes similar abortions in cattle. Recent reports of abortions in dairy cows in the eastern U.S. are believed to be linked to ingestion of Cupressus (Monterey Cypress) ingestion. Again, losses associated with the abortion do not take into account other losses such as forage, management, costs of replacing lost cows, cow deaths, added veterinary care etc. In the south western U.S. broom snakeweed (Gutierrezia spp.) causes annual estimated losses of over $40 million to the livestock industry. These losses include toxicoses, abortion, retained placenta and lost production in cattle, sheep and goats and loss of forage production from snakeweed
invasion. In addition, because of its allelopathic nature, broom snakeweed aggressively invades grass lands decreasing available good forage, compromising native plant biodiversity and inhibiting optimum utilization of many of these rangelands. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? National Program 108, Food Safety: Through chemical analysis the Poisonous Plant Research Lab has identified many individual toxins and metabolites in lupines, pine trees, juniper spp., broom snakeweed etc. Physiologic/toxic activity has been determined in target species and rodent models. An ELISA has been developed for some of the toxins and certain metabolites found in pine needles and juniper . Some of these toxins have been screened in vitro for biological activity using oocyte/embryo cultures. Using ELISA and sophisticated chemistry technology, the toxin, isocupressic acid, (ICA, the abortifacient toxin in pines/junipers)
and selected metabolites of ICA were identified in blood serum of pregnant cows. Absorption and disappearance of ICA and metabolites will be determined in blood. An ELISA was developed for the teratogenic steroidal alkaloids cyclopamine and jervine in blood and animal tissues. Teratogenic and toxic alkaloids have been identified in lupines and other plants and absorption and elimination profiles in animal serum are being investigated. This research will provide information and guidelines to improve the quality of feeds for livestock, enhance product quality for consumers of animal products and better prognosis of disease. Management strategies and diagnostic tools (ELISA's) are being developed to identify toxicoses from pine needles, lupines and other teratogenic plants. This research will provide information to assist in the prevention of plant induced birth defects. National Programs 205, Rangeland, Pasture and Forage: Over the last 3 years grazing studies have been done to
determine why and when cattle graze pine needles and lupines. Forage quality, environmental factors and animal grazing behavior have been monitored to determine conditions when cattle graze these plants. This research will enhance grazing strategies to prevent these birth defects from recurring and prevent the associated economic losses to the livestock industry and enhance the economic base of ranchers and the communities in which they live. 4. What were the most significant accomplishments this past year? A 3 year grazing study was conducted in eastern Washington to determine when cows eat lupine (Lupinus leucophyllus) and correlate consumption to the critical period of gestation which causes the crooked calf syndrome. In all 3 years, cows started eating lupine around the first of July after cheatgrass had matured, and the other forbs were grazed out or matured (Lupine consumption ranged from 17 to 25% of diets). This period of consumption overlaps the critical period of
gestation (40-70 day of pregnancy) if the traditional breeding seasons begins May 1, and cattle should be removed from lupine areas during this period. Broom snakeweed is an invasive native plant that crowds out desirable species and causes abortions if cows consume it during the last trimester of gestation. We attempted to positively condition non-pregnant heifers to preferentially graze snakeweed as a biological tool to suppress it in the sagebrush steppe plant community in northern Utah. The heifers were fed snakeweed then given starch as a positive energy feedback to enhance their acceptance of snakeweed. Conditioning was successful in the pen as starch-fed heifers consumed more snakeweed than untreated controls, but the heifers would not graze snakeweed in the field until all other forage was consumed. Then the positively conditioned heifers consumed more snakeweed than the controls. Cattle in high (7.5 on 9 point scale) and low (3.5) body condition were fed green pine
needles free choice during a 2week period. Cattle in low (i.e., thin) body condition ate more (P=0.001) pine needles than did fat cows (5.5 g pine needles/kg body weigt vs. 1.0 g/kg, respectively). 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. * Research at the Poionsous Plant Research Lab on Veratrum californicum and lupines began in the early 1950's and were identified as the cause of the 'malformed lamb syndrome' and 'crooked calf disease' respectively. * Toxins and teratogens from Veratrum and lupines were identified and characterized. * Developed ELISA's for detection of ICA and it's sera metabolites and cyclopamine and jervine (teratogens from Veratrum) using polyclonal antibodies. Using these tools and simple extraction techniques, rapid screening of biological samples for the presence of these toxins and or their metabolites was accomplished. * A collaborative study with the University of Idaho on absorption
and elimination of lupine alkaloids in sheep differing in body condition showed that thin sheep more rapidly absorbed toxic alkaloids than did fat sheep, and conversely thin sheep showed slower elimination of alkaloids compared to fat animals. * Grazing trials with fat and thin cattle indicated that thin cattle initially ate more lupine than did fat animals in eastern Washington. Eventually all animals ate substantial quantities of lupine, but thin animals ate more lupine at times, thus the risk of intoxication is particularly high for thin animals compared to cattle in higher body condition. *Isolated and identified the abortifacient toxin in pine needles as a labdane resin acid, isocupressic acid, ICA. Major serum metabolites were identified as dihydroagathic acid, agathic acid, imbricatoloic acid, and tetrahydroagathic acid. Maximum serum metabolites occured between 24 and 48 hours after consumption of pine needles. * Isocupressic acid was identified in Cupressus macrocarpa
(monterey cypress), Pinus contorta (lodgepole pine), Juniperus communis (common juniper). In subsequent feeding trials lodgepole pine and common juniper were demonstrated to be abortifacient. * Twenty-three tree and shrub species from throughout the western and southern states were analyzed for ICA. Significant levels (>0.5% dry weight) were detected in Pinus ponderosa, P. jefferyi, P. contorta (lodgepole pine), Juniperus scopulorum (Rocky Mountain juniper) and J. communis (common juniper). Similar toxins have been identified in broom snakeweed but the putative abortifacient compound has not yet been identified. Identification and measurement of the toxic chemicals in the pine needles can provide useful information for the range scientist and ranchers in formulating management practices to prevent losses. * Grazing and pen studies examined conditioned food aversions as a potential tool to keep cattle from consuming pine needles. Cattle were successfully averted to green needles
initially, but the aversion extinguished as cattle began consuming dry needles and then green needles. In nutrition trials, low energy status initially influenced cows to eat more needles but as cows gained experience with needles this treatment effect diminished; supplemental protein did not result in increased pine needle consumption. *Using in vitro fertilization techniques, oocytes and embryos were cultured in the presence of isocupressic acid (the pine needle toxin) or various steroidal alkaloid teratogens including cyclopamine from Veratrum californicum, a synthetic cyclopamine derivative and three steroidal alkaloid teratogens from Solanum. Steroidal alkaloid teratogens from Veratrum and Solanum spp. inhibited embryo growth and development in vitro when introduced in the culture media at different concentrations. Isocupressic acid enhanced oocyte maturation and early embryo growth and development. ICA-treated embryos were transferred to recipient cows, and while pregnancy
progressed in a normal fashion, 2 of 5 calves were born dead and necropsy revealed an identical cardiovascular anomaly in both calves. While the increase in embryo development induced by ICA may have significant utility in IVF systems, further evaluation is required. * Grazing studies conducted in eastern Washington in cooperation with Washington State University from 2001 through 2003 determined that cattle begin to graze lupine when associated forages dry out or are depleted from the plant community. Cattlemen can use this basic information to modify their breeding programs and or grazing strategies to reduce the risk of subsequent lupine-induced birth defects. 6. What do you expect to accomplish, year by year, over the next 3 years? Year 1: 1) Conduct a grazing study to determine if increasing grazing pressure by overgrazing will force cattle to graze lupine. 2) Continue lupine population study to evaluate effects of weather on lupine population density. 3) Continue ecology
study to determine long-term impact of fire and grazing on snakeweed populations. 4) Profile the diterpene acids present in broomsnake weed plants. 5) Screen broom snakeweed plant material and serum from cattle after broom snakeweed consumption using the recently developed enzyme-linked immuno assay procedure to identify possible structurally related compounds that are the abortifacient components. 6) Continue investigation of immunologic activity of ICA in an effort to develop a vaccine in cows. 7) Individual quinolizidine and piperidine alkaloids will be isolated from lupines and other teratogenic plants for comparative toxicity/teratogenicity testing. Year 2. 1) Continue to explore scale up synthesis of ICA metabolites. 2) Large scale synthesis of ICA metabolites for animal trials will be done. 3) Continue investigating the toxic and abortifacient activity of broom snakeweed. 4) Lupine grazing studies on other sites and under other environmental conditions in eastern
Washington to further define when cattle will graze lupine will be done. 5) A summary bulletin will be prepared with current information and recommendation for management of lupine grazing on the scablands of eastern Washington. 6) Continue grazing studies on the relationship between forage availability/quality and pine needle and lupine consumption. Year 3. 1) Continue screening broom snakeweed plants for abortifacient compounds. 2) Continue investigation of abortifacient metabolites in the serum of cattle fed pine needles or common juniper. 3) Scale up synthesis of the same compounds for animal feeding trials. 4 ) Grazing studies will continue on the Channel Scablands of eastern Washington to determine when and why cattle graze lupines. 5) Management strategies will be developed and recommendations will be given to ranchers in the Scabland region to reduce calf losses. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology
likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Management to reduce livestock losses from poisonous plants. Influence of drought on losses to toxic plants. Presented at the Arizona Strip Workshop, March 26, 2003, Kanab Utah, and March 27, 2003, St. George, Utah. Sponsored by Arizona and Utah Extension Services. The information presented at these workshop is available to ranchers and is being implemented in many grazing programs. There is considerable interest from scientists doing research on human, using cyclopamine, the teratogen from Veratrum californicum and its ability to block the effects of a certain family of genes known as sonic hedgehog. Purified crystalline cyclopamine has been prepared from V. californicum and shared with nine different research organizations to be used in a number of different in vitro studies designed to investigate the
role of the hedgehog pathway. Examples include the study of specific genes in the sonic hedgehog pathway in the cerebellum; effects of pesticides on crustacean development and shell formation; mechanism of action of the anti-tumor drug tamoxifen; the regulation of neural tissue repair; studies on craniofacial neural crest cells; and hedgehog pathway activity in medulloblastoma growth. Information has been transferred to other scientists through publications in peer-reviewed journals and to scientists, land managers, extension agents and ranchers through popular publications, seminars, work shops, e-mail and telephone conversations. This information is now available for ranchers use to develop grazing strategies to reduce losses. As new information is made available, producer meetings are held to transfer new information and technologies. The major constraints to adoption of current technology include changing environmental conditions, forage availability and acceptance of new
management programs. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). "Cattle Consumption of lupine in the scablands of Eastern Washington" by Michael Ralphs, Kip Panter, Jim Pfister, Clive Gay and Ernie Motteram. Presented at Society for Range Management annual meeting, Feb 2, 2003, CAsper WY. Panter, K.E. Presented seminar on toxic and teratogenic plants at 'Plant Interactions and the Effect of Global Warming on Alpine Biodiversity' workshop in Kazbegi, Republic of Georgia, June 30-July 6, 2003l. Pfister, J.A. Presented two workshops at the Arizona Strip, March 26, 2003, Kanab Utah, and March 27, 2003, St. George, Utah. Sponsored by Arizona and Utah Extension Services. Results of lupine research have frequently been discussed with extension agents and ranchers in many western states.
Impacts
(N/A)
Publications
- Panter, K.E., James, L.F., Wang, .S., Gardner, D.R., Gaffield Jr, W.P., Molyneux, R.J., Stegelmeier, B.L., Bunch, .T. 2004. Screening poisonous plant toxins for cytotoxicity using bovine embryos produced by in vitro fertilization techniques. Poisonous Plants Symposium Proceedings.
- LEE, S.T., PANTER, K.E., GAFFIELD JR, W.P., STEGELMEIER, B.L. DEVEELOPMENT OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY FOR THE PLANT TERATOGENS CYCLOPAMINE AND JERVINE. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY. 2003.
- LEE, S.T., GARDNER, D.R., GARROSSIAN, M., PANTER, K.E., SERREQI, A.N., SCHOCH, T.K., WIERENGA, T.L. DEVELOPMENT OF ENZYME-LINKED IMMUNOSORBENT ASSAYS FOR ISOCUPRESSIC ACID AND SERUM METABOLITES OF ISOCUPRESSIC ACID. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY. 2003.
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Progress 10/01/01 to 09/30/02
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Grazing of ponderosa pine needles causes abortion/premature parturition, retained placentas and endometritis in pregnant cattle resulting in substantial economic losses to cattle producers in the western U.S. and Canada. Lodgepole pine and Monterey Cypress also cause abortion in cattle. Ponderosa pine needle calf losses were reported as recently as the spring of 2001 near Laramie, Wyoming. Similar losses occur in the southwestern U.S. when cattle, sheep and goats graze broom snakeweed. Lupines cause poisoning and "crooked calf syndrome" in cattle on many ranges in the western U.S. Extensive losses to cattle producers in the Channel Scablands of eastern Washington State have occurred the last few years with catastrophic losses occurring in 1997. Other teratogenic plants also contribute to the overall losses from poisonous plants to the livestock community. Research on these problems is
being addressed using a multidisciplinary approach requiring research on five main objectives: C Isolation and characterization of abortifacient, toxic and teratogenic compounds in pine needles, broom snakeweed, lupine and other teratogenic plant, and identifying metabolic or detoxification derivatives found in the animal that exhibit abortifacient or teratogenic activity. C Describe physiologic activity in animals and determine serum profiles of absorption, metabolism and clearance of abortifacient and teratogenic compounds and potentially active metabolites. C Define the mechanism of action or how pine needles/broom snakeweed cause the abortions and how teratogenic plants cause birth defects. Describe pathophysiology, endocrine, immunologic and morphologic changes associated with these conditions. C Develop conjugates or immunogenic derivatives to the abortifacient and teratogenic compounds and evaluate potential vaccines, antitoxins and toxin scavengers and determine what
treatments can be applied to reduce losses from premature or deformed offspring and retained placentas. C Determine animal, environmental, and management factors influencing consumption of ponderosa, broom snakeweed and lupines and develop strategies to reduce losses. 2. How serious is the problem? Why does it matter? Pine needles (PN) cause an estimated loss of over $20 million annually to cattle producers in the western U.S. Pine needles cause abortions/premature births and retained placenta in cattle when ingested during the last trimester of pregnancy. Also, ponderosa pines are rapidly invading grass lands on forest boundaries reducing grass production and inhibiting maximum efficiency of many of these ranges. The problem is pervasive in the western United States and Canada where cattle graze in pine forests. Lodgepole pine has recently been shown to cause abortion in cattle and losses have been reported in western Canada. Undoubtedly, other pines that contain the
abortifacient toxin isocupressic acid (ICA) contribute to losses on a regional, national and international basis. In Australia and New Zealand the tree Cupressus macrocarpa causes similar abortions in cattle. Recent reports of abortions in dairy cows in the eastern U.S. are believed to be linked to ingestion of Cupressus (Monterey Cypress) ingestion. Broom snakeweed (Gutierrezia spp.) causes annual estimated losses of over $40 million to the livestock industry in the southwestern U.S. Losses include toxicoses, abortion, retained placenta in cattle, sheep and goats and loss of forage production from snakeweed invasion. In addition, because of its allelopathic nature, broom snakeweed aggressively invades grass lands decreasing available good forage, plant biodiversity and inhibits optimum utilization of many of these rangelands. Lupines and other teratogenic plants cause large losses annually to the livestock industry in the western U.S. Catastrophic losses from lupines have
recently occurred in the Channel Scablands of eastern Washington State and Central Oregon with the most recent loss reporting over 4,000 calves in Adams County Washington ($1.7 million) in 1997. This estimate does not take into account many of the increased management costs associated with such a large loss, for example increased culling, lost grazing opportunities, fencing, additional feed, etc. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? National Program 108, Food Safety: Research at the Poisonous Plant Research Lab has identified many toxins and certain first phase metabolites responsible for pine needle-induced abortion and plant induced birth defects. An ELISA has been developed that will assist in diagnosis and detection of isocupressic acid (the pine needle abortifacient), certain ICA metabolites and the teratogenic steroidal alkaloids cyclopamine and jervine in animal tissues. Many teratogenic alkaloids
have been identified in lupines and other plants and absorption and elimination profiles in animal serum are being investigated. This research will provide information and guidelines to reduce losses from abortifacient and teratogenic plants. Management strategies and diagnostic tools (ELISA's) are being developed to identify toxicoses from pine needles, lupines and other teratogenic plants. This research will enhance diagnosis and treatment of animal diseases, especially those associated with abortion and birth defects. This research will enhance utilization of grazing lands conserving the Nation's natural resource base and balance production efficiency with environmental quality. 4. What was your most significant accomplishment this past year? ELISA's were developed for ICA and its sera metabolites using polyclonal antibodies. One assay is specific to isocupressic acid while the other three assays show cross reactivity with agathic acid, dihydroagathic acid and tetrahydroagathic
acid. Using this assay, the absorption into the serum and elimination into the urine of three ICA metabolites was measured in a cow dosed orally with common juniper. Similarly, an ELISA was developed to detect and measure two significant teratogenic steroidal alkaloids in Veratrum californicum, cyclopamine and jervine. Using these tools and simple extraction techniques, rapid screening of biological samples for the presence of these toxins and/or their metabolites was accomplished. There is little information on the effects of body condition (i.e., thin vs. fat animals) on ingestion and metabolism of toxic plants. A collaborative study with the University of Idaho on absorption and elimination of lupine alkaloids in sheep differing in body condition showed that thin sheep more rapidly absorbed toxic alkaloids than did fat sheep, and conversely thin sheep showed slower elimination of alkaloids compared to fat animals. These results suggest that thin animals will be intoxicated to
a greater extent than will animals in better body condition. Subsequently, grazing trials with fat and thin cattle indicated that thin cattle initially ate more lupine than did fat animals in eastern Washington. Eventually all animals ate substantial quantities of lupine, but thin animals ate more lupine at times, thus the risk of intoxication is particularly high for thin animals compared to cattle in higher body condition. We do not know when and why cattle graze lupine. However, a grazing study conducted in eastern Washington in cooperation with Washington State University in 2001 and again 2002 determined that cattle begin to graze lupine when associated forages dry out or are depleted from the plant community. Cattlemen can use this basic information to modify their grazing strategies to reduce the risk of subsequent lupine-induced birth defects. 5. Describe your major accomplishments over the life of the project, including their predicted or actual impact? Significant
accomplishments over the life of the project include: the isolation and identification of the abortifacient toxin in pine needles (a labdane resin acid, isocupressic acid, ICA). After extensive extractions and fractionation experiments, the toxins in pine needles were identified as isocupressic acid (ICA) and two ICA derivatives, succinyl and acetyl ICA. Laboratory studies determined that succinyl and acetyl ICA are converted in the rumen of cattle to ICA. In the rumen and after absorption, ICA is further metabolized and rumen and serum metabolites of ICA were identified. The major metabolites were dihydroagathic acid, agathic acid, imbricatoloic acid, and tetrahydroagathic acid. Maximum serum metabolite concentrations were identified and occur between 24 and 48 hours after consumption of pine needles. The next phase of the work will be to determine abortifacient significance of or metabolic action of these major metabolites. Compounds belonging to the same class of compounds as
ICA have been identified in broom snakeweed but the exact abortifacient toxin has not been identified yet. Broom snakeweed also contains many other compounds that are overtly toxic to cattle. Isocupressic acid was identified in Cupressus macrocarpa (monterey cypress), Pinus contorta (lodgepole pine), and Juniperus communis (common juniper). In subsequent feeding trials lodgepole pine and common juniper were demonstrated to be abortifacient. Twenty-three tree and shrub species from throughout the western and southern states were analyzed for ICA. Significant levels (>0.5% dry weight) were detected in Pinus ponderosa, P. jefferyi, P. contorta (lodgepole pine), Juniperus scopulorum (Rocky Mountain juniper) and J. communis (common juniper). Similar toxins have been identified in broom snakeweed but the putative abortifacient compound has not yet been identified. Identification and measurement of the toxic chemicals in the pine needles can provide useful information for the range
scientist and ranchers in formulating management practices to prevent losses. Ponderosa and lodgepole pine needles, common juniper and Cupressus macrocarpa (an abortifacient tree from New Zealand containing ICA) were also analyzed for the presence of vasoactive lipids including myristate and laurate esters of 1,14-tetradecanediol and 1,12-dodecanediol. These vasoactive lipids have been proposed as possible abortifacient components in pine needles. Concentration of these vasoactive lipids were 0.028%, 0. 023%, 0.001% and ND for ponderosa, lodgepole, common juniper and C. macrocarpa, respectively. This research confirms that isocupressic acid is the abortifacient compound and suggests that these lipids with vasoactive properties in vitro are not involved in pine needle-induced abortion in cattle. Grazing and pen studies examined conditioned food aversions as a potential tool to keep cattle from consuming pine needles. Cattle were successfully averted to green needles initially, but
the aversion extinguished as cattle began consuming dry needles and then green needles. In nutrition trials, low energy status initially influenced cows to eat more needles but as cows gained experience with needles this treatment effect diminished; supplemental protein did not result in increased pine needle consumption. An isocupressic acid-protein conjugate was prepared that elicited an immune response in cows and sheep. The sheep antisera was used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection and measurement of isocupressic acid and metabolites. This assay will be used to measure isocuprssic acid such as agathic acid, dihydroagathic acid, and tetrahydroagathic acid in blood samples. The assay will also be used to measure isocupressic acid, and succinyl and acetyl isocupressic acid in plant material. A serum metabolite of isocupressic acid was synthesized to confirm its structure and presence in the serum of cattle after consumption of Ponderosa pine
needles. This metabolite could be an important diagnostic marker for pine needle abortion as it has a longer resident time in the serum than the other reported metabolites. The synthetic metabolites will be useful in preparing immuno-conjugates for development of ELISA analytical methods for diagnostics and detection in tissues and for potential vaccine development. Using in vitro fertilization techniques, oocytes and embryos were cultured in the presence of isocupressic acid (the pine needle toxin) or various steroidal alkaloid teratogens including cyclopamine from Veratrum californicum, a synthetic cyclopamine derivative and three steroidal alkaloid teratogens from Solanum. The alkaloid teratogens inhibited embryo growth and development when introduced at different concentrations. Conversely, isocupressic acid enhanced oocyte maturation and early embryo growth and development. ICA-treated embryos were transferred to recipient cows, and while pregnancy progressed in a normal
fashion, 2 of 5 calves were born dead and necropsy revealed an identical cardiovascular anomaly in both calves. While the increase in embryo development induced by ICA may have significant utility in IVF systems, further evaluation is required. Significant accomplishments over the life of the project include: the isolation and identification of the abortifacient toxin in pine needles (a labdane resin acid, isocupressic acid, ICA). After extensive extractions and fractionation experiments, the toxins in pine needles were identified as isocupressic acid (ICA) and two ICA derivatives, succinyl and acetyl ICA. Laboratory studies determined that succinyl and acetyl ICA are converted in the rumen of cattle to ICA. In the rumen and after absorption, ICA is further metabolized and rumen and serum metabolites of ICA were identified. The major metabolites were dihydroagathic acid, agathic acid, imbricatoloic acid, and tetrahydroagathic acid. Maximum serum metabolite concentrations were
identified and occur between 24 and 48 hours after consumption of pine needles. The next phase of the work will be to determine abortifacient significance of or metabolic action of these major metabolites. Compounds belonging to the same class of compounds as ICA have been identified in broom snakeweed but the exact abortifacient toxin has not been identified yet. Broom snakeweed also contains many other compounds that are overtly toxic to cattle. Isocupressic acid was identified in Cupressus macrocarpa (monterey cypress), Pinus contorta (lodgepole pine), and Juniperus communis (common juniper). In subsequent feeding trials lodgepole pine and common juniper were demonstrated to be abortifacient. Twenty-three tree and shrub species from throughout the western and southern states were analyzed for ICA. Significant levels (>0.5% dry weight) were detected in Pinus ponderosa, P. jefferyi, P. contorta (lodgepole pine), Juniperus scopulorum (Rocky Mountain juniper) and J. communis (common
juniper). Similar toxins have been identified in broom snakeweed but the putative abortifacient compound has not yet been identified. Identification and measurement of the toxic chemicals in the pine needles can provide useful information for the range scientist and ranchers in formulating management practices to prevent losses. Ponderosa and lodgepole pine needles, common juniper and Cupressus macrocarpa (an abortifacient tree from New Zealand containing ICA) were also analyzed for the presence of vasoactive lipids including myristate and laurate esters of 1,14-tetradecanediol and 1,12-dodecanediol. These vasoactive lipids have been proposed as possible abortifacient components in pine needles. Concentration of these vasoactive lipids were 0.028%, 0. 023%, 0.001% and ND for ponderosa, lodgepole, common juniper and C. macrocarpa, respectively. This research confirms that isocupressic acid is the abortifacient compound and suggests that these lipids with vasoactive properties in
vitro are not involved in pine needle-induced abortion in cattle. Grazing and pen studies examined conditioned food aversions as a potential tool to keep cattle from consuming pine needles. Cattle were successfully averted to green needles initially, but the aversion extinguished as cattle began consuming dry needles and then green needles. In nutrition trials, low energy status initially influenced cows to eat more needles but as cows gained experience with needles this treatment effect diminished; supplemental protein did not result in increased pine needle consumption. An isocupressic acid-protein conjugate was prepared that elicited an immune response in cows and sheep. The sheep antisera was used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection and measurement of isocupressic acid and metabolites. This assay will be used to measure isocuprssic acid such as agathic acid, dihydroagathic acid, and tetrahydroagathic acid in blood samples. The assay will
also be used to measure isocupressic acid, and succinyl and acetyl isocupressic acid in plant material. A serum metabolite of isocupressic acid was synthesized to confirm its structure and presence in the serum of cattle after consumption of Ponderosa pine needles. This metabolite could be an important diagnostic marker for pine needle abortion as it has a longer resident time in the serum than the other reported metabolites. The synthetic metabolites will be useful in preparing immuno-conjugates for development of ELISA analytical methods for diagnostics and detection in tissues and for potential vaccine development. Using in vitro fertilization techniques, oocytes and embryos were cultured in the presence of isocupressic acid (the pine needle toxin) or various steroidal alkaloid teratogens including cyclopamine from Veratrum californicum, a synthetic cyclopamine derivative and three steroidal alkaloid teratogens from Solanum. The alkaloid teratogens inhibited embryo growth and
development when introduced at different concentrations. Conversely, isocupressic acid enhanced oocyte maturation and early embryo growth and development. ICA-treated embryos were transferred to recipient cows, and while pregnancy progressed in a normal fashion, 2 of 5 calves were born dead and necropsy revealed an identical cardiovascular anomaly in both calves. While the increase in embryo development induced by ICA may have significant utility in IVF systems, further evaluation is required. Significant accomplishments over the life of the project include: the isolation and identification of the abortifacient toxin in pine needles (a labdane resin acid, isocupressic acid, ICA). After extensive extractions and fractionation experiments, the toxins in pine needles were identified as isocupressic acid (ICA) and two ICA derivatives, succinyl and acetyl ICA. Laboratory studies determined that succinyl and acetyl ICA are converted in the rumen of cattle to ICA. In the rumen and
after absorption, ICA is further metabolized and rumen and serum metabolites of ICA were identified. The major metabolites were dihydroagathic acid, agathic acid, imbricatoloic acid, and tetrahydroagathic acid. Maximum serum metabolite concentrations were identified and occur between 24 and 48 hours after consumption of pine needles. The next phase of the work will be to determine abortifacient significance of or metabolic action of these major metabolites. Compounds belonging to the same class of compounds as ICA have been identified in broom snakeweed but the exact abortifacient toxin has not been identified yet. Broom snakeweed also contains many other compounds that are overtly toxic to cattle. Isocupressic acid was identified in Cupressus macrocarpa (monterey cypress), Pinus contorta (lodgepole pine), and Juniperus communis (common juniper). In subsequent feeding trials lodgepole pine and common juniper were demonstrated to be abortifacient. Twenty-three tree and shrub species
from throughout the western and southern states were analyzed for ICA. Significant levels (>0.5% dry weight) were detected in Pinus ponderosa, P. jefferyi, P. contorta (lodgepole pine), Juniperus scopulorum (Rocky Mountain juniper) and J. communis (common juniper). Similar toxins have been identified in broom snakeweed but the putative abortifacient compound has not yet been identified. Identification and measurement of the toxic chemicals in the pine needles can provide useful information for the range scientist and ranchers in formulating management practices to prevent losses. Ponderosa and lodgepole pine needles, common juniper and Cupressus macrocarpa (an abortifacient tree from New Zealand containing ICA) were also analyzed for the presence of vasoactive lipids including myristate and laurate esters of 1,14-tetradecanediol and 1,12-dodecanediol. These vasoactive lipids have been proposed as possible abortifacient components in pine needles. Concentration of these vasoactive
lipids were 0.028%, 0. 023%, 0.001% and ND for ponderosa, lodgepole, common juniper and C. macrocarpa, respectively. This research confirms that isocupressic acid is the abortifacient compound and suggests that these lipids with vasoactive properties in vitro are not involved in pine needle-induced abortion in cattle. Grazing and pen studies examined conditioned food aversions as a potential tool to keep cattle from consuming pine needles. Cattle were successfully averted to green needles initially, but the aversion extinguished as cattle began consuming dry needles and then green needles. In nutrition trials, low energy status initially influenced cows to eat more needles but as cows gained experience with needles this treatment effect diminished; supplemental protein did not result in increased pine needle consumption. An isocupressic acid-protein conjugate was prepared that elicited an immune response in cows and sheep. The sheep antisera was used to develop an enzyme-linked
immunosorbent assay (ELISA) for the detection and measurement of isocupressic acid and metabolites. This assay will be used to measure isocuprssic acid such as agathic acid, dihydroagathic acid, and tetrahydroagathic acid in blood samples. The assay will also be used to measure isocupressic acid, and succinyl and acetyl isocupressic acid in plant material. A serum metabolite of isocupressic acid was synthesized to confirm its structure and presence in the serum of cattle after consumption of Ponderosa pine needles. This metabolite could be an important diagnostic marker for pine needle abortion as it has a longer resident time in the serum than the other reported metabolites. The synthetic metabolites will be useful in preparing immuno-conjugates for development of ELISA analytical methods for diagnostics and detection in tissues and for potential vaccine development. Using in vitro fertilization techniques, oocytes and embryos were cultured in the presence of isocupressic acid
(the pine needle toxin) or various steroidal alkaloid teratogens including cyclopamine from Veratrum californicum, a synthetic cyclopamine derivative and three steroidal alkaloid teratogens from Solanum. The alkaloid teratogens inhibited embryo growth and development when introduced at different concentrations. Conversely, isocupressic acid enhanced oocyte maturation and early embryo growth and development. ICA-treated embryos were transferred to recipient cows, and while pregnancy progressed in a normal fashion, 2 of 5 calves were born dead and necropsy revealed an identical cardiovascular anomaly in both calves. While the increase in embryo development induced by ICA may have significant utility in IVF systems, further evaluation is required. 6. What do you expect to accomplish, year by year, over the next 3 years? Year 1: (1) Profile the diterpene acids present in broomsnake weed plants. (2) Collect broom snakeweed from different populations for chemical characterization.
(3) Conduct nutritional studies of supplemental feeding regimes designed to reduce consumption of pine needles by grazing cattle. (4) Grazing studies on the Channel Scablands of Washington State will be done to determine why and when cattle graze lupines. (5) Pen studies will be conducted using native cattle to determine absorption and elimination profiles of the lupine alkaloids, especially the teratogen anagyrine. (6) Continue studies on lupine and body condition in cattle and /or sheep. (7) Broom snakeweed is toxic and generally an unpalatable plant. We understand little about the conditions that lead grazing livestock to eat the plant; nonetheless, pregnant animals often abort from consuming snakeweed. Trials are underway to determine if broom snakeweed has aversive properties to livestock in order to shed light on factors important in consumption of snakeweed. Year 2. (1) Screen broom snakeweed plant material and serum from cattle after broom snakeweed consumption using
the recently developed enzyme- linked immuno assay procedure to identify possible structurally related compounds that are the abortifacient components in broomsnake weed plants. (2) Explore scale-up synthesis of ICA metabolites (3) Continue investigation of immunologic activity of ICA and continue evaluation of vaccine in cows. (4) Continue to define nutritional status of cows and subsequent propensity to graze pine needles and broom snakeweed. (5) Large scale synthesis of ICA metabolites for animal trials will be done. (6) Continue investigating the toxic and abortifacient activity of broom snakeweed. (7) Lupine grazing studies on other sites and under other environmental conditions in eastern Washington to further define when cattle will graze lupine will be done. (8) Continue grazing studies on relationship between forage availability/quality and pine needle and lupine consumption. (9) Continue grazing studies and aversion conditioning studies with cattle grazing pine
needles. Year 3. (1) Continue screening broom snakeweed plants for abortifacient compounds. (2) Continue investigation of abortifacient metabolites in the serum of cattle fed pine needles or common juniper and continue scale up synthesis of the same compounds for animal feeding trials. (3) Lupine studies will continue on the Channel Scablands of eastern Washington to determine when and why cattle graze lupines. (4) Management strategies will be developed and recommendations will be given to ranchers in the Scabland region to reduce calf losses. (5) Individual quinolizidine and piperidine alkaloids will be isolated from lupines and other teratogenic plants for comparative toxicity/teratogenicity testing. 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? Information has been transferred to
other scientists through publications in peer-reviewed journals and to scientists, land managers, extension agents and ranchers through popular publications, seminars, work shops, e- mail and telephone conversations. This information is now available for ranchers to use to develop grazing strategies to reduce losses. As new information is made available, producer meetings are held to transfer new information and technologies. The major constraints to adoption of current technology include changing environmental conditions, forage availability and acceptance of new management programs. 8. List your most important publications and presentations, and articles written about your work (NOTE: this does not replace your review publications which are listed below) Research results on lupine grazing studies and toxicology information were presented to ranchers of the Adams County WA cattlemans Association on 18 June, 2002 in Ritzeville, WA. Discussions were presented by Dr. Lynn F. James,
Dr. James A. Pfister and Dr. Michael H. Ralphs of our laboratory and by cooperators from Washington State University. Presented "management of toxic plants" to the Idaho Weed Association and the Western Wyoming Weed Association. Presented to Animal Science and Veterinary Science faculty and students, University of Sao Paulo, Brazil "Poisonous Plant Research at PPRL" and "Behavioral effects of toxic plant ingestion in livestock.
Impacts
(N/A)
Publications
- Wang, S., Panter, K.E., Gaffield, W., Evans, R.C. and Bunch, T.D. Effects of three natural occurring steroidal glycoalkaloids from potatoes (Solanum tuberosum) on in vitro bovine embryo development. Western Section, American Society Animal Science, 2002, v. 53, p. 147-150.
- Pfister, J.A., Provenza, F.D., Panter, K.E., Stegelmeier, B.L. and Launchbaugh, K.L. Risk management to reduce livestock losses from toxic plants. Journal Range Management, 2002, v. 55, p.291-300.
- Panter, K.E., James, L.F., Gardner, D.R., Ralphs, M.H., Pfister, J.A., Stegelmeier, B.L. and Lee, S.T. Influence of management strategies on reproductive losses to poisonous plants. Journal Range Management, 2002, v. 55, p. 301-308.
- Garrossian, M., Gardner, D.R., Panter, K.E., James, L.F. Preparation of tetrahydroagathic acid: a serum metabolite of isocupressic acid, a cattle abortifacient in ponderosa pine. Journal of Agriculture and Food Chemistry, 2002, v. 50(8), p. 2235-2240.
- Wang, S., Panter, K.E., Gardner, D.R., Evans, R.C. and Bunch, T.D. Effects of isocupressic acid (ICA), the abortifacient toxin from ponderosa pine needles, on in vitro bovine oocyte maturation and subsequent embryo development. Western Section, American Society Animal Science, 2002, v. 53, p. 257-259.
- Ralphs, M.H. and Sanders, K.D. Population cycles of broom snakeweed in the Colorado Plateau and Snake River Plains. Journal Range Management, v. 55, p. 406-411.
- Panter, K.E., Wang, S. Gaffield, W., James, L.F., Evans, R.C. and Bunch, T. D. Effects of cyclopamine, a Veratrum alkaloid, and its synthetic analog cyclopamine-4-ene-3-one on in vitro bovine oocyte maturation and subsequent embryo development. Western Section, American Society Animal Science, 2002, v. 53, p. 383-386.
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