Excess consumption of selenium (Se) accumulator plants can result in selenium intoxication. The objective of the study reported here was to compare the acute toxicosis caused by organic selenium (selenomethionine) found in plants with that caused by the supplemental, inorganic form of selenium (sodium selenite). Lambs were orally administered a single dose of selenium as either sodium selenite or selenomethionine and were monitored for 7 days, after which they were euthanized and necropsied. Twelve randomly assigned treatment groups consisted of animals given 0, 1, 2, 3, or 4 mg of Se/kg of body weight as sodium selenite, or 0, 1, 2, 3, 4, 6, or 8 mg of Se/kg as selenomethionine. Sodium selenite at dosages of 2, 3, and 4 mg/kg, as well as selenomethionine at dosages of 4, 6, and 8 mg/kg resulted in tachypnea and/or respiratory distress following minimal exercise. Severity and time to recovery varied, and were dose dependent. Major histopathologic findings in animals of the high-dose groups included multifocal myocardial necrosis and pulmonary alveolar vasculitis with pulmonary edema and hemorrhage. Analysis of liver, kidney cortex, heart, blood, and serum revealed linear, dose-dependent increases in selenium concentration. However, tissue selenium concentration in selenomethionine-treated lambs were significantly greater than that in lambs treated with equivalent doses of sodium selenite. To estimate the oxidative effects of these selenium compounds in vivo, liver vitamin E concentration also was measured. Sodium selenite, but not selenomethionine administration resulted in decreased liver vitamin E concentration. Results of this study indicate that the chemical form of the ingested Se must be known to adequately interpret tissue, blood, and serum Se concentrations.

Anabasine occurring in wild tree tobacco (Nicotiana glauca) and anabaseine occurring in certain animal venoms are nicotinic receptor agonist toxins. Anabasine lacks the imine double bond of anabaseine; the two possible enantiomers of anabasine occur in N. glauca. A comparision of the relative potencies of S- and R-anabasine has not been previously reported. We separated the enantiomers of anabasine by reaction of the racemic N. glauca natural product with 9-fluorenylmethoxycarbonyl-L-alanine (Fmoc-L-Ala-OH) to give diastereomers, which were separated by preparative reversed phase HPLC. The S- and R-anabasine enantiomer fractions were then obtained by Edman degradation. A mouse bioassay was used to determine the relative lethalities of S- and R-enriched anabasine enantiomers. The intravenous LD50 of the (+)-R-anabasine rich fraction was 11 +/- 1.0 mg/kg and that of the (-)-S-anabasine-rich fraction was 16 +/- 1.0 mg/kg. The LD50 of anabaseine was 0.58 +/- 0.05 mg/kg. Anabaseine was significantly more toxic in the mouse bioassay than S-anabasine (27-fold) and R-anabasine (18-fold). The relative agonistic potencies of these three alkaloids on human fetal nicotinic neuromuscular receptors were of the same rank order: anabaseine>>R-anabasine>S-anabasine.

Certain lupines (Lupinus spp.) contain alkaloids that cause contracture-type skeletal birth defects and cleft palate (‘‘crooked calf syndrome’’) when consumed by cows during the 40th to 70th day of gestation. The objective of this study was to determine when cattle graze velvet lupine (Lupinus leucophyllus Dougl.) during its phenological development, and whether this period overlaps the critical period of gestation. Grazing studies were conducted in 2001, 2002, and 2003 in the same 100 ha pasture in eastern Washington. A second objective was addressed in 2001 to determine if cows with crooked calves consumed more lupine than cows with normal calves. Five mature Hereford cows with crooked calves at their side and 6 mature Hereford cows with normal calves grazed together for the summer. There was no difference (P = 0.17) in the amount of lupine consumed between groups, and all cows consumed some lupine. In 2002, 10 3-year-old Hereford cows with normal calves were used, and in 2003, 8 of the same cows from the 2002 study with normal calves were used. In all 3 years, cows started consuming lupine in July and August after annual grasses dried and annual forbs matured. Lupine is a deep-rooted perennial that remained green and succulent longer into the summer than the associated forages. Concentration of the teratogenic alkaloid anagyrine declined as lupine seeds shattered in late June and early July. Lupine consumption occurred during the critical period of gestation in 2 of the 3 years, but no crooked calves were produced. Apparently, the cows did not ingest sufficient amounts of anagyrine over the susceptible period of time to produce crooked calves. The management recommendation for this site is to restrict access to lupine during July when cattle begin to graze lupine and anagyrine levels may still be relatively high. Once the seeds shatter, toxicity greatly declines.

We hypothesize that there are inherent differences in the levator veli palatini (LVP) of non-clefted (NP) and clefted palates (CP) that become more pronounced with age that could impact the timing and outcome of cleft palate repair. The LVP from 15 goats were studied: 1) 2-month-old (MO) goats with NP (N=5); 2) 2 MO goats with CP (N=4); 3) 14 MO goats with NP (N=3); and 4) 14 MO goats with CP (N=3). H&E and myosin ATPase stains were used to measure fiber cross-sectional areas (CSAs) and for fiber typing. 2 MO is an age analogous to when human cleft palate repairs are performed. In the 2 MO group, there were no differences in muscle fiber CSAs or composition between NP and CP goats. CSAs of the 14 MO CP muscle fibers (1974 +/− 939 μm2) were larger (p<0.05) than the areas of the 14 MO NP muscle fibers (1651 +/− 680 μm2). The CSAs between the two age groups were different with larger CSA in the 14 MO goats (p<0.05). The fiber types in the CP muscle were not different between the ages studied. However, there were differences in muscle fiber types between 14 MO CP and NP LVP muscles; CP muscles contained both Type 1 (29.3 +/− 10.6%) and Type 2 (70.7 +/− 10.6%) muscle fibers while the NP muscles contained exclusively Type 1 muscle fibers (100%). Our study demonstrates that the CP muscle fibers increase in CSA and fast fiber type with age; indicating a maladaptive response in comparison with NP musculature. T32 GM008616-06A1; P30 AG13283.

Locoweed poisoning has been reported in wildlife, but it is unknown whether mule deer (Odocoileius hemionus) are susceptible. In areas that are heavily infested with locoweed, deer and elk (Cervus elaphus nelsoni) have developed a spongiform encephalopathy, chronic wasting disease (CWD). Although these are distinct diseases, no good comparisons are available. The purpose of this study was to induce and describe chronic locoweed poisoning in deer and compare it with the lesions of CWD. Two groups of four mule deer were fed either a complete pelleted ration or a similar ration containing 15% locoweed (Oxytropis sericea). Poisoned deer lost weight and developed a scruffy, dull coat. They developed reluctance to move, and movement produced subtle intention tremors. Poisoned deer had extensive vacuolation of visceral tissues, which was most severe in the exocrine pancreas. Thyroid follicular epithelium, renal tubular epithelium, and macrophages in many tissues were mildly vacuolated. The exposed deer also had mild neuronal swelling and cytoplasmic vacuolation that was most obvious in Purkinje cells. Axonal swelling and dystrophy was found in many white tracts, but it was most severe in the cerebellar peduncles and the gracilis and cuneate fasciculi. These findings indicate that deer are susceptible to locoweed poisoning, but the lesions differ in severity and distribution from those of other species. The histologic changes of locoweed poisoning are distinct from those of CWD in deer; however, the clinical presentation of locoweed poisoning in deer is similar. Histologic and immunohistochemical studies are required for a definitive diagnosis.

Ammodendrine (1) was found to occur as a mixture of enantiomers in two different collections of plants identified as Lupinus formosus. The ammodendrine fraction was reacted in a peptide coupling reaction with 9-fluorenylmethoxycarbonyl-L-alanine (Fmoc-L-Ala-OH) to give diastereomers, which were separated by preparative HPLC. The pure D- and L-ammodendrine enantiomers were then obtained by Edman degradation. Optical rotation measurements revealed that the D- and L-enantiomers had optical rotations of [alpha]24D +5.4 and -5.7, respectively. D- and L-N-methylammodendrine enantiomers were synthesized from the corresponding ammodendrine enantiomers, and their optical rotations established as [alpha]23D +62.4 and -59.0, respectively. A mouse bioassay was used to determine the difference in toxicity between these two pairs of naturally occurring enantiomers. The LD50 of (+)-D-ammodendrine in mice was determined to be 94.1 +/- 7 mg/kg and that of (-)-L-ammodendrine as 115.0 +/- 7 mg/kg. The LD50 of (+)-D-N-methylammodendrine in mice was estimated to be 56.3 mg/kg, while that of (-)-L-N-methylammodendrine was determined to be 63.4 +/- 5 mg/kg. These results establish the rotation values for pure ammodendrine and N-methylammodendrine and indicate that there is little difference in acute murine toxicity between the respective enantiomers.

The objective of this study is to determine the effects of intermittent locoweed (O. sericea) poisoning on the development of clinical and histological lesions. Results suggest that sheep and possibly cattle may ingest locoweeds for short periods, 5 days or less, if allowed withdrawal periods of 7 to 14 days without developing behavioural or functional lesions.

OBJECTIVE To compare plasma disposition of alkaloids after lupine challenge in cattle that had given birth to calves with lupine-induced arthrogryposis and cattle that had given birth to clinically normal calves and determine whether the difference in outcome was associated with differences in plasma disposition of anagyrine. ANIMALS 6 cows that had given birth to calves with arthrogryposis and 6 cows that had given birth to clinically normal calves after being similarly exposed to lupine during pregnancy. PROCEDURES Dried lupine (2 g/kg) was administered via gavage. Blood samples were collected before and at various time points for 48 hours after lupine administration. Anagyrine, 5,6-dehydrolupanine, and lupanine concentrations in plasma were measured by use of gas chromatography. Plasma alkaloid concentration versus time curves were generated for each alkaloid, and pharmacokinetic parameters were determined for each cow. RESULTS No significant differences in area under the plasma concentration versus time curve, maximum plasma concentration, time to reach maximum plasma concentration, and mean residence time for the 3 alkaloids were found between groups. CONCLUSIONS AND CLINICAL RELEVANCE Because no differences were found in plasma disposition of anagyrine following lupine challenge between cattle that had given birth to calves with arthrogryposis and those that had not, our findings do not support the hypothesis that between-cow differences in plasma disposition of anagyrine account for within-herd differences in risk for lupine-induced arthrogryposis.

Research designed to isolate and identify the bioactive compounds responsible for the toxicity of plants to livestock that graze them has been extremely successful. The knowledge gained has been used to design management techniques to prevent economic losses, predict potential outbreaks of poisoning, and treat affected animals. The availability of these compounds in pure form has now provided scientists with tools to develop animal models for human diseases, study modes of action at the molecular level, and apply such knowledge to the development of potential drug candidates for the treatment of a number of genetic and infectious conditions. These advances are illustrated by specific examples of biomedical applications of the toxins of Veratrum californicum (western false hellebore), Lupinus species (lupines), and Astragalus and Oxytropis species (locoweeds).

α-Solanine and α-chaconine are two naturally occurring steroidal glycoalkaloids in potatoes (Solanum tuberosum), and solanidine-N-oxide is a corresponding steroidal aglycone. The objective of this research was to screen potential cyto-toxicity of these potato glycoalkaloids using bovine oocyte maturation, in vitro fertilization techniques and subsequent embryonic development as the in vitro model. A randomized complete block design with four in vitro oocyte maturation (IVM) treatments (Experiment 1) and four in vitro embryo culture (IVC) treatments (Experiment 2) was used. In Experiment 1, bovine oocytes (n = 2506) were matured in vitro in medium supplemented with 6μM of α-solanine, α-chaconine, solanidine-N-oxide or IVM medium only. The in vitro matured oocytes were then subject to routine IVF and IVC procedures. Results indicated that exposure of bovine oocytes to the steroidal glycoalkaloids during in vitro maturation inhibited subsequent pre-implantation embryo development. Potency of the embryo-toxicity varied between these steroidal glycoalkaloids. In Experiment 2, IVM/IVF derived bovine embryos (n = 2370) were cultured in vitro in medium supplemented with 6μM of α-solanine, α-chaconine, solanidine-N-oxide or IVC medium only. The results showed that the pre-implantation embryo development is inhibited by exposure to these glycoalkaloids. This effect is significant during the later pre-implantation embryo development period as indicated by fewer numbers of expanded and hatched blastocysts produced in the media containing these alkaloids. Therefore, we conclude that in vitro exposure of oocytes and fertilized ova to the steroidal glycoalkaloids from potatoes inhibits pre-implantation embryo development. Furthermore, we suggest that ingestion of Solanum species containing toxic amounts of glycoalkaloids may have negative effects on pre-implantation embryonic survival.

Several species of lupine (Lupinus spp.) are poisonous to livestock, producing death in sheep and "crooked calf disease" in cattle. Range livestock cope with poisonous plants through learned foraging strategies or mechanisms affecting toxicant disposition. When a toxic plant is eaten, toxicant clearance may be influenced by the animal's nutritional and/or physiological status. This research was conducted to determine whether differences in body condition or short-term nutritional supplementation of sheep altered the disposition of lupine alkaloids given as a single oral dose of ground silvery lupine (Lupinus argenteus) seed. Ewes in average body condition (ABC, n = 9) and low body condition (LBC, n = 10) received a single dose of ground lupine seeds including pods (8.5 g/kg BW) via gavage on the first day of the experiment, and were then randomly assigned to one of two nutritional supplement treatments. Blood samples were taken 0 to 60 h after dosing to compare blood alkaloid concentration and to evaluate alkaloid absorption and elimination profiles. Concentrations of total alkaloid and anagyrine, 5,6 dehydrolupanine, lupanine, and alkaloid E were measured in serum. These four alkaloids constituted 78 and 75% of the total alkaloid concentration in serum for LBC vs. ABC groups, respectively. Initial analysis indicated that short-term supplementation had no effect on alkaloid disposition, and supplementation was removed from the statistical model. The highest concentration of total alkaloids was observed 2 h after dosing. Overall, serum total alkaloid and anagyrine levels (area under the curve) were higher (P < 0.01) for sheep in the LBC group. Serum peak concentrations of total alkaloid and anagyrine were higher in LBC vs. ABC groups (P < 0.05). Serum elimination of anagyrine, unknown alkaloid E, and lupanine was decreased in LBC vs. ABC treatments (P < 0.05). These results demonstrate that body condition is important in the disposition of lupine alkaloids; however, further research is needed to determine the potential benefit, if any, that short-term nutritional supplementation might have on alkaloid disposition.

Larkspur poisoning sporadically kills from 5 to 15% of the cattle on North American mountain rangelands. Of the 40 different diterpenoid larkspur alkaloids, the one that is thought to be responsible for much of the toxicity has been identified as methyllycaconitine (MLA). Little is known of MLA toxicokinetics or excretion. The purpose of this study was to further characterize the clinical effects of MLA toxicity in mice and determine the toxicokinetics of MLA excretion. Eight groups of mice were dosed intravenously with 2.0 mg/kg of BW of MLA, killed, and necropsied at 0, 1, 2, 5,10,15, 30, and 60 min after injection. Treated animals were reluctant to move, trembled, and developed dyspnea, muscular twitches, and convulsions. Within several minutes, the clinical signs abated and behavior slowly returned to normal over approximately 20 min. At necropsy serum, brain, liver, kidney, and skeletal muscle were collected and frozen. Blood and tissues were extracted and analyzed for MLA with HPLC and electron spray mass spectrometry. Blood MLA elimination followed a normal biphasic redistribution and excretion pattern (r = 0.99) with a K of elimination of 0.0376 and half-life of 18.4 min. Other tissues had similar clearance rates. These data indicate the MLA is rapidly distributed and excreted. In mice, the clinical effects of poisoning seem to affect the central nervous system, causing dyspnea and "explosive" muscular twitches and convulsions. Because livestock commonly eat larkspur at subclinical doses, they are likely to have larkspur alkaloids in many tissues. These results suggest that animals exposed to larkspur should rapidly excrete MLA (within several hours) and that the residues in animal tissues are not likely to be a problem if animals are given several days to allow toxin clearance.

The consumption of ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), common juniper (Juniperus communis), and Monterey cypress (Cupressus macrocarpa) causes abortions in pregnant cattle. Recent studies have identified isocupressic acid (1) as the primary abortificient compound in these plants. In vitro and in vivo studies using rumen and blood have shown isocupressic acid (1) is rapidly metabolized to agathic acid (3), dihydroagathic acid (4), and tetrahydroagathic acid (5). Rapid and sensitive diagnostic techniques are needed to identify poisoned animals, to study toxicokinetics, and to elucidate the mechanism of isocupressic acid-induced abortion in cattle. In this study, four competitive inhibition enzyme-linked immunosorbent assays for isocupressic acid and its sera metabolites were developed using polyclonal antibodies. One assay is specific to 1, whereas the other three assays show cross-reactivity to 3-5 in addition to 1. The assay specific to 1 had a limit of detection of 44.1 pg. The other assays which demonstrated cross-reactivity to the isocupressic acid blood metabolites also had comparably low limits of detection. One assay was used to follow the absorption and elimination profile of isocupressic acid metabolites in both cow serum and urine after oral dosage of a cow with common juniper.

Veratrum californicum was responsible for large losses of sheep grazing high mountain ranges in central Idaho in the 1950s. Veratrum induces various birth defects including the cyclopic-type craniofacial defect (monkey-faced lambs) that is specifically induced in lambs after pregnant ewes grazed the plant on the 14th day of gestation. The steroidal alkaloids cyclopamine (1) and jervine (2) were isolated from Veratrum and shown to be primarily responsible for the malformations. Cyclopamine (1) and jervine (2) are potent teratogens that inhibit Sonic hedgehog (Shh) signaling during gastrulation-stage embryonic development, producing cyclopia and holoprosencephaly. Although losses to the sheep industry from Veratrum are now relatively infrequent, occasional incidents of toxicoses and craniofacial malformations are still reported in sheep and other species. However, the benefits to biomedical research using cyclopamine (1) as a tool to study human diseases have greatly expanded. A competitive inhibition enzyme-linked immunosorbent assay (ELISA) to detect and measure cyclopamine (1) and jervine (2) was developed using polyclonal antibodies produced in ewes. The limits of detection of the assay were 90.0 and 22.7 pg for cyclopamine (1) and jervine (2), respectively. This assay was used for the detection and measurement of cyclopamine (1) spiked into sheep blood. The simple extraction-ELISA methods developed in this study demonstrate the potential of using these techniques for the rapid screening of biological samples to detect the presence and concentration of cyclopamine (1) and jervine (2) and will be beneficial to pharmacological studies and livestock diagnostics.

The purpose of this study was to determine whether larkspur toxins conjugated to protein carriers would promote active immunity in mice. Mice were injected with several larkspur toxin-protein conjugates or adjuvant alone to determine whether the resulting immunological response altered animal susceptibility to methyllycaconitine, the major toxic larkspur alkaloid. Although vaccinations increased the calculated lethal dose 50% (LD50) for intravenous methyllycaconitine toxicity, overlapping confidence intervals did not provide evidence of differences between the vaccinated and control groups. In the lycoctonine conjugate (LYC)-vaccinated group, mouse survival was related (P = 0.001) to serum titers for methyllycaconitine doses up to 4.5 mg/kg of body weight. When mice withlow antibody titers were removed from the vaccinated groups in which titer was related to survival, the recalculated LD50 estimates were 20% greater than the LD50 of the control group. However, the 95% confidence intervals of the recalculated LD50 groups overlapped with the control groups. Overall, these results suggest that vaccination altered methyllycaconitine toxicity in mice and that vaccination may be useful in decreasing the effects of larkspur toxins in animals. Additional studies are warranted to continue development of potential larkspur vaccines for livestock.

The objective of this study is to evaluate the effects of animal age on pyrrolizidine alkaloid (PA) toxicity, especially the effects of PAs on neonatal animals. Poisoned 3- and 6-week-old pigs developed clinical signs that were more severe than other age groups. Results suggest that weanling pigs near 3-6 weeks of age are most susceptible to PA poisoning.

Systematic approaches to taxonomic classifications of the tall larkspur spp. have been developed using traditional chemical methods to profile alkaloids, comparison of relative toxicity of individual alkaloids, plant morphology/taxonomy and molecular genetics. Using these methods (papers published in this series) toxicology of three distinct species of tall larkspurs including Delphinium glaucum, Delphinium barbeyi and Delphinium occidentale is described. Tall larkspurs (Delphinium spp.) continue to be the most serious cause of cattle losses on mountain rangelands in the western US. Over 40 norditerpenoid alkaloids have been reported in species of larkspurs and toxicology data on 25 of these have been reported by the authors. These alkaloids can be classified into three general types based on their structural characteristics and toxicity: the N-(methylsuccinyl) anthranoyl lycoctonine (MSAL)-type, having high toxicity; the lycoctonine-type, with moderate toxicity; and the 7,8-methylenedioxylycoctonine (MDL)-type, of low toxicity. The structural importance of the methylsuccinimido anthranilic acid ester group at the C18 position is evident in the high toxicity of MSAL alkaloids, particularly methyllycaconitine (MLA), Nudicauline (NUD) and 14-deacetylnudicauline (14-DAN). Other structural aspects of these alkaloids such as the C14 functionality are also important, as demonstrated by the reduced toxicity of barbinine. MLA is the alkaloid of most importance in toxicity of larkspurs on mountain rangelands because of its prevalence in most larkspurs and high toxicity. While NUD and 14-DAN also possess high toxicity, they are relatively minor components in few larkspur species (generally the plains and low larkspurs), but when present at concentrations approaching 1 mg/g dry weight they contribute significantly to overall toxicity. Deltaline (DLT) is often found in high concentrations in many larkspurs but because of low toxicity, its contribution to larkspur poisoning in the field is relatively minor and it will probably not cause toxicosis in the absence of the MSAL-type alkaloids.

Poisonous plants that impair normal reproductive functions in livestock include Veratrum californicum Durand, lupines, ponderosa pine (Pinus ponderosa Dougl.), broom snakeweed (Gutierrezia sarothrae (Pursh) Britt. Rusby), locoweeds (Astragalus and Oxytropis spp.), selenium-containing forages, phytoestrogenic plants, endophyte-infected grasses and others. In this review we focus on lupines, locoweeds and ponderosa pine needles to demonstrate the broad and diverse effects that poisonous plants have on reproduction. Certain lupines (Lupinus spp.) contain quinolizidine and piperidine alkaloids that are fetotoxic and when grazed by pregnant cattle during specific stages of gestation induce skeletal birth defects and cleft palate, "crooked calf disease". Poison-hemlock (Conium maculatum) and some Nicotiana spp. contain similar alkaloids and induce identical birth defects in cattle, pigs, goats and sheep when ingested at certain stages of gestation. Locoweeds (species of the Astragalus and Oxytropis genera containing the indolizidine alkaloid swainsonine) interfere with most processes of reproduction when grazed for prolonged periods of time. Animals can recover normal reproductive function if withdrawn from locoweed grazing before severe poisoning occurs. While most animals may recover reproductive function, permanent neurological deficits may preclude normal reproductive behavior. Ponderosa and lodgepole pine needles (Pinus spp.) cause abortion in cattle when grazed during the last trimester of gestation. The specific chemical constituents responsible for the abortions belong to a class of compounds called labdane resin acids, including isocupressic acid (ICA), succinyl ICA, and acetyl ICA. Basic management recommendations to reduce reproductive losses to poisonous plants include: (1) keep good records; (2) know what poisonous plants grow on ranges and understand their effects; (3) develop a management plan to provide for alternate grazing in poisonous plant-free pastures during critical times; (4) provide for balanced nutrition, including protein, energy, minerals and vitamins; (5) maintain a good herd health program; (6) integrate an herbicide treatment program to reduce poisonous plant populations or to maintain clean pastures for alternate grazing; and, (7) manage the range for maximum forage production.

Risk of livestock losses to poisonous plants can be reduced on many ranges through prudent management based on application of existing knowledge. Poisonous plants can be categorized using both acceptability to livestock and a plant's toxic potential. Acceptability encompasses forage qualities such as taste and chemistry (i.e., nutrient and toxin concentrations) and postingestive feedback from an animal's daily and long-term (e.g., body condition) nutritional and toxicological state. Toxic potential reflects aspects of plant chemistry, including seasonal or other changes in concentration or functionality of the toxin(s), and type of toxicity (i.e., acute or chronic). Persistent livestock losses to poisonous plants may indicate that ranges are over-grazed or improperly managed. Aggressive management schemes that employ high stocking rates and grazing intensities may yield greater returns, but may also increase risk if poisonous plants are present. Plants may be ranked according to toxicity and acceptability. Six interrelated categories of plants are discussed: 1) always toxic and acceptable to livestock; 2) always toxic and not acceptable; 3) always toxic and acceptable at certain times; 4) toxic only at certain times and acceptable to livestock; 5) toxic at certain times and unacceptable; and 6) toxic at certain times and acceptable at certain times. Each category involves differing risk and uncertainty. Within this management matrix, strategies for dealing with specific poisonous plants can be customized depending on how much and when the plant is eaten by livestock, and when the plant is most toxic.

Isocupressic acid (1) was used to synthetically prepare a mixture of (8S,13R,S)-labda-15,19-dioic acid (tetrahydroagathic acid) (5) via a two-step oxidation procedure followed by hydrogenation of the double bonds at C13 and C8. Reduction of the C8,17 double bond was stereospecific producing only the 8S isomer and confirmed by the nOe interaction between the resulting C17 and C20 methyl groups. The 13R and 13S isomers of 5 were separated and analyzed by HPLC/MS, and (13S)-tetrahydroagathic acid was isolated and identified by comparison to a standard prepared by hydrogenation of naturally occurring (13S)-dihydroagathic acid (4). (13R,S)-dihydroagathic acid was prepared by selective sodium metal-catalyzed hydrogenation of the C13,14 allylic double bond of agathic acid (3). The prepared compounds were then used as standards to confirm the presence of 4 and 5 and their respective 13R and 13S isomers in bovine serum samples. Tetrahydroagathic acid was shown to be the only metabolite detected in serum samples taken from a suspected cattle abortion case submitted for diagnosis; and, thus, 5 could be a valuable diagnostic marker for pine needle-induced abortions.