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It is well understood that water hemlock tubers are highly toxic to animals and to humans. However, this is the first time that immature seed from (Cicuta maculata) has been implicated in livestock poisoning. Nine mature Hereford cows from a herd of 81 died in northwestern Utah after ingesting immature seed heads of water hemlock (Cicuta maculata) in late summer. No obvious signs of poisoning were reported as all nine were found dead near the banks of the stream where water hemlock was found. Upon discovery of the dead cows, the remaining 72 cows were immediately removed from the pasture and no further losses occurred. Field necropsy of 3 of the dead cows and follow-up serology and histopathological examination of selected tissues did not identify any bacterial or viral causes. History of ingestion of large quantities of water hemlock seed, the acute nature of the deaths, chemical comparison of seed with toxic tubers and follow-up mouse bioassay testing supported the diagnosis of water hemlock poisoning. Seed heads collected from the neighboring pasture upstream and across the fence from the poisoned cattle and tubers collected from grazed plants were chemically analyzed and found to contain cicutoxin, and high levels of two cicutol-like derivatives (cicutol-#1 and #2) as well as other unidentified polyacetylene compounds. Seeds and tubers from suspected plants were semi-quantified and compared to archive samples of highly toxic tubers used in previous experiments. The immature hemlock seed contained less cicutoxin (0.01 times), but 9.5 and 22.5 times more cicutol-#1 and cicutol-#2 respectively, compared to the archive sample. Tubers from the grazed plants contained 4.6 times more cicutoxin and 9.8 and 18.8 times more cicutol-#1 and cicutol-#2 respectively, compared to the archive sample. Mouse bioassays with water extracts of immature seed and tubers from grazed plants demonstrated both were highly toxic and of greater toxicity when compared to archived sample., (Copyright © 2010. Published by Elsevier Ltd.)

Teratogenic alkaloids are found in many species of plants including Conium maculatum L., Nicotiana glauca, Nicotiana tabaccum, and multiple Lupinus spp. Fetal musculoskeletal defects produced by alkaloids from these plants include arthrogyropisis, scoliosis, torticollis, kyposis, lordosis, and cleft palate. A pharmacodynamic comparison of the alkaloids ammodendrine, anabasine, anabaseine, anagyrine, and coniine in SH-SY5Y cells and TE-671 cells was made. These alkaloids and their enantiomers were more effective in depolarizing TE-671 cells which express the human fetal-muscle type nicotinic acetylcholine receptor (nAChR) relative to SH-SY5Y cells which predominately express autonomic nAChRs. The rank order of potency in TE-671 cells was: anabaseine>(+)-anabasine>(-)-anabasine > (+/-)-anabasine>anagyrine>(-)-coniine > (+/-)-coniine>(+)-coniine>(+/-)-ammodendrine>(+)-ammodendrine. The rank order potency in SH-SY5Y cells was: anabaseine>(+)-anabasine>(-)-coniine>(+)-coniine>(+)-ammodendrine>anagyrine>(-)-anabasine>(+/-)-coniine>(+/-)-anabasine>(-)-ammodendrine. The actions of these alkaloids at nAChRs in both cell lines could be distinguished by their maximum effects in depolarizing cell membrane potential. The teratogenic action of these compounds may be related to their ability to activate and subsequently desensitize nAChRs.

Objective: To determine the contribution of 7,8-methylenedioxylycoctonine (MDL)-type alkaloids to the toxic effects of tall larkspur (Delphinium spp) consumption in cattle., Animals: Sixteen 2-year-old Angus steers., Procedures: Plant material from 3 populations of tall larkspur that contained different concentration ratios of MDL-type-to-N-(methylsuccinimido) anthranoyllycoctonine (MSAL)-type alkaloids was collected, dried, and finely ground. For each plant population, a dose of ground plant material that would elicit similar clinical signs of toxicosis in cattle after oral administration was determined on the basis of the plants' MSAL-type alkaloid concentration. Cattle were treated via oral gavage with single doses of ground plant material from each of the 3 populations of tall larkspur; each animal underwent 1 to 3 single-dose treatments (> or = 21-day interval between treatments). Heart rate was recorded immediately before (baseline) and 24 hours after each larkspur treatment., Results: Tall larkspur populations with a lower MDL-type-to-MSAL-type alkaloid concentration ratio required a greater amount of MSAL-type alkaloids to cause the expected clinical signs of toxicosis (including increased heart rate) in cattle., Conclusions and Clinical Relevance: Results indicated that the typically less toxic MDL-type alkaloids contributed in a significant manner to the toxic effects of tall larkspur in steers. Consequently, both the concentration of MSAL-type alkaloids and the total concentration of MSAL- and MDL-type alkaloids should be determined when assessing the relative toxicity of tall larkspur populations. These results provide valuable information to determine the risk of toxicosis in cattle grazing on tall larkspur-infested rangelands.

Freshly ground Utah juniper [Juniperus osteosperma (Torr.) Little] bark was given via gavage at a dosage of 2.3 kg per cow twice daily to three pregnant cows starting on day 255 of gestation. All three cows aborted the calves after 4, 5 and 6 days of treatment. A fourth cow was dosed with Utah juniper needles and this cow calved early on day 268 of gestation with complications consistent with pine needle abortion. Chemical analysis of Juniperus osteosperma bark identified the major diterpene acid as the labdane acid known as agathic acid. Agathic acid was measured in the bark at a concentration of 1.5% (dry weight basis). Analysis of sera samples obtained from treated cows found detectable quantities of agathic acid, dihydroagathic acid and tetrahydroagathic acid, which are known serum metabolites of the abortifacient compound isocupressic acid. Based on the high incidence of induced abortion and detection of known metabolites in affected animals, the labdane acid known as agathic acid is considered to be an abortifacient compound in late-term pregnant cattle.

Objective: To describe the simple elimination kinetics of methyllycaconitine (MLA) and deltaline and evaluate the heart rate response in cattle following oral administration of larkspur., Animals: 5 healthy Angus steers that were habituated to metabolism crates., Procedures: Tall larkspur (Delphinium barbeyi) in the early flowering stage was collected, dried, and ground. Each steer received a single dose of larkspur that was equivalent to 10.4 mg of MLA/kg and 11.0 mg of deltaline/kg via oral administration. Steers were housed in metabolism crates during a 96-hour period following larkspur administration; heart rate was monitored continuously, and blood samples were collected periodically for analysis of serum MLA and deltaline concentrations as well as assessment of pharmacokinetic parameters., Results: No overt clinical signs of poisoning developed in any steer during the experiment. Mean +/- SE heart rate reached a maximum of 79.0 +/- 5.0 beats/min at 17 hours after larkspur administration. Serum MLA concentration was correlated directly with heart rate. Mean times to maximal serum concentration of MLA and deltaline were 8.8 +/- 1.2 hours and 5.0 +/- 0.6 hours, respectively. Mean elimination half-life values for MLA and deltaline were 20.5 +/- 4.1 hours and 8.2 +/- 0.6 hours, respectively., Conclusions and Clinical Relevance: Following larkspur administration in 5 healthy steers, maximum serum concentrations of MLA and deltaline were detected within 10 hours, and changes in serum MLA concentration and heart rate were correlated. Results indicated that cattle that have consumed larkspur will eliminate 99% of MLA and deltaline from serum within 144 hours.

Cyclopamine, a steroidal alkaloid, from the plant Veratrum californicum is teratogenic, causing a range of different birth defects. The critical window for cyclopamine-induced synophthalmia formation has been reported to be gestational day (GD) 14. The objectives of this study were to better describe cyclopamine-induced craniofacial deformities, to better define the window of susceptibility to synophthalmia formation, and to characterize cyclopamine toxicokinetics in sheep. Ewes were dosed i.v. with purified cyclopamine for toxicokinetic analysis. Another four groups of ewes were dosed orally twice daily with 0.88 g/kg of V. californicum on GD 13, 14 or 15 or consecutively on GD days 13-15. Pregnancy and pre-partum fetal malformations were determined by ultrasound imaging on GD 60. At parturition lambs were assessed for gross malformations. The elimination half-life of cyclopamine in ewes was determined to be 1.1 +/- 0.1 h. The rapid clearance of cyclopamine indicates that ingestion of V. californicum must occur during a very narrow window for synophthalmia formation to occur. Ewes dosed with V. californicum on GD 13 or 14 had lambs with various craniofacial malformations including cyclopia, maxillary dysplasia and mandibular micrognathia. Ewes dosed on GD 15 delivered normal lambs. Ewes dosed consecutively on GD 13-15 were not pregnant at GD 60 and Veratrum-induced embryonic death was assumed to be the cause. Interestingly, lambs with cyclopia were smaller, under-developed and appeared premature even though their twin appeared fully developed. Initial evaluations suggest this was due to placental dysplasia., (2009 by John Wiley & Sons, Ltd.)

Although the mechanism of action for larkspur alkaloids has been described, little information is available on the variation of the physiological response of individual animals to larkspur alkaloids. Anecdotal observations and pilot studies in cattle indicate that there is animal-to-animal variation in response to a debilitating dose of larkspur alkaloids. The objective of this study was to determine whether there is variation in susceptibility of different strains of mice to larkspur alkaloid toxicosis and to identify factors responsible for the variation that could then be used as a model for studies in cattle. The acute toxicity of methyllycaconitine (MLA) in 9 different inbred strains of mice was compared. The rank order, from most to least susceptible, was A/J>B10>FVB>BALB/c>C57Bl/6>NZW>C3H>DBA>129. The calculated LD(50) ranged from 3.3+/-0.2 to 5.8+/-0.8 mg/kg of BW. The toxicokinetic profiles of MLA in the susceptible A/J strain and the more resistant 129 strain were compared to determine whether their differences in susceptibility were due to differences in their ability to eliminate MLA. The differences in toxicokinetic variables observed did not explain the differences in susceptibility. The protein expression of various nicotinic acetylcholine receptor (nAChR) subunits was also compared between the more resistant 129 strain and the susceptible A/J strain. The 129 strain of mice had twice the amount of alpha7 nAChR subunit expression as the A/J strain, which was in direct proportion to the approximately 2-fold difference in LD(50). There was also a significant difference (P<0.05) in expression of the alpha3 and alpha5 nAChR subunits between the 129 and A/J strains, with the 129 strain having a greater expression in each case. These data suggest that the increased susceptibility of the A/J mice could be due to a reduced expression of nAChR subunits. Similar analyses need to be made in cattle to determine whether there is a difference between breeds in susceptibility to larkspur poisoning and to identify the factors that regulate their susceptibility to larkspur poisoning. This information would be useful for livestock producers in their breeding, culling, and grazing management programs to reduce or prevent larkspur poisoning on rangelands.

OBJECTIVE-To determine whether larkspur-derived N-(methylsuccinimido) anthranoyllycoctonine (MSAL)-type alkaloids alter heart rate and electrically evoked electromyographic (eEMG) response of the external anal sphincter (EAS) in cattle and whether these effects can be reversed by acetylcholinesterase inhibitors. ANIMALS-12 beef heifers and 4 cows. PROCEDURES-3 or 4 heifers were used in 1 or 2 of 7 dose-response experiments; heart rate and EAS eEMG response were assessed before and 24 hours after oral treatment with larkspur (doses equivalent to 0.5 to 15 mg of MSAL-type alkaloids/kg). In 3 subsequent experiments, 3 heifers (1 of which was replaced with another heifer in the control experiment) each received 10 mg of MSAL-type alkaloids/kg and were injected IV with physostigmine (0.04 mg/kg), neostigmine (0.04 mg/kg), or saline (0.9% NaCl) solution 24 hours later, prior to assessment. Additionally, EAS eEMG response was measured in 4 cows before and after epidural administration of 2% lidocaine hydrochloride. RESULTS-Larkspur-treated heifers developed dose-related increases in heart rate and decreases in EAS eEMG response. Twenty-four hours after administration of MSAL-type alkaloids, neostigmine decreased heart rate but did not affect eEMG response, whereas physostigmine did not affect heart rate but caused a 2-fold increase in eEMG response. In cows, epidural anesthesia did not alter eEMG response, suggesting that transdermal stimulation of the EAS pudendal innervation did not occur. CONCLUSIONS AND CLINICAL RELEVANCE-In cattle, cardiac effects and muscle weakness or loss of EAS eEMG response induced by larkspur-derived MSAL-type alkaloids were reversed by neostigmine or physostigmine, respectively. Treatment with anticholinesterase inhibitors may alter the clinical effects of larkspur poisoning in cattle.

Lupines are common plants on the rangelands in the western United States. Lupines contain alkaloids that can be toxic and teratogenic causing congenital birth defects (crooked calf disease). One such lupine, Lupinus sulphureus, occurs in parts of Oregon, Washington, and British Columbia. Specimens of L. sulphureus from field collections and herbaria were evaluated taxonomically and by chemical means. A total of seven distinct alkaloid profiles and the individual alkaloids associated with each profile were identified. Each alkaloid profile was unique in its geographical distribution and its potential risk to livestock. In conclusion, taxonomic classification is not sufficient to determine risk, as chemical characterization of the alkaloids must also be performed.

We determined whether cows in low (LBC) or high body condition (HBC) would consume different amounts of green pine needles (Pinus ponderosa). Cows (mature; open Hereford and Hereford x Angus) were fed a maintenance basal diet (alfalfa pellets) for Exp. 1 and 2; during Exp. 3 and 4, cows were fed high-protein and high-energy diets, respectively. Experiment 5 was a grazing study on rangeland during winter in South Dakota; diets were determined by using bite counts. Mean BCS (1 = emaciated, 9 = obese) was 7.5 for HBC cows and <4.0 for LBC cows during the experiments. During Exp. 1, LBC cows consumed more (P = 0.001) pine needles than did HBC cows (5.5 +/- 0.25 vs. 1.0 +/- 0.14 g/kg of BW daily, respectively). During Exp. 2, there was a day x treatment interaction (P = 0.001) as LBC cows consumed variable, but greater, amounts of pine needles than did HBC cows (3.7 +/- 0.19 vs. 1.3 +/- 0.12 g/kg of BW daily, respectively). When fed a high-protein/low-energy diet, LBC cows ate more (P = 0.04) pine needles than did HBC cows. When fed a low-protein/high-energy diet, there was a day x treatment interaction (P = 0.001) because LBC cows consumed more pine needles than did HBC cows for the first 3 d of the study, and then consumption by LBC animals decreased during the last 4 d. These experiments suggest that the protein:energy ratio may be an important factor in the ability of cows to tolerate terpenes, and that cows were not able to sustain an increased quantity of needle consumption on a low-protein diet. During the 25-d grazing study, there was a day x treatment interaction (P = 0.001) as LBC animals selected more pine needles (up to 25% of daily bites) on some days compared with HBC cows. Weather influenced pine needle consumption because pine needle bites by LBC cows were related (r(2) = 0.60; P = 0.001) to days of greater snow depth and lower minimum daily temperatures. Both LBC and HBC cows increased selection of pine needles from trees during cold, snowy weather, but the magnitude of the increase was greater for LBC cows. The LBC cows consumed more pine needles than did HBC cows in all experiments, except when cows were fed a low-protein diet. This study indicates that both body condition and protein intake are important factors in pine needle consumption.

Several species of lupine (Lupinus spp.) are toxic to livestock, causing death losses in sheep and cattle but more commonly crooked calf disease in pregnant range cows. The major toxic alkaloids in lupine are of the quinolizidine alkaloid group and include the teratogen anagyrine, which is primarily responsible for crooked calf disease. Lupines also contain teratogenic piperidine alkaloids including ammodendrine. Previous work in sheep has shown that lupine alkaloid clearance may be influenced by the animal's physiological status. Therefore, the purpose of this study was to determine if differences in body condition of cattle would alter the absorption and elimination of anagyrine or ammodendrine given in a single oral dose as Lupinus leucophyllus or Lupinus sulphureus, respectively. Mature non-lactating cows in low body condition (LBC, n = 4) and high body condition (HBC, n = 4) received a single dose of dry ground lupine plant (2.0 g/kg of BW) via oral gavage. Lupinus leucophyllus (anagyrine) was dosed first; then after 21 d the same animals were dosed with L. sulphureus (ammodendrine). Blood samples were taken via jugular venipuncture 0 to 60 h after dosing. Serum anagyrine and ammodendrine concentrations were evaluated. The concentration of anagyrine was greater (P = 0.001) in the HBC group and peaked 2 h after dosing versus 12 h in LBC cows. Similarly for ammodendrine, the alkaloid concentration peaked at 3 h after dosing for the HBC group compared with 6 h for the LBC group (P = 0.001). Area under the curve tended to differ (P

Larkspur plants contain numerous norditerpenoid alkaloids, which include the 7,8-methylenedioxylycoctonine (MDL)-type alkaloids and the N-(methylsuccinimido)anthranoyllycoctonine (MSAL)-type alkaloids. The MSAL-type alkaloids are generally much more toxic (typically >20 times). Toxicity of many tall larkspurs, such as Delphinium barbeyi, has been attributed to its large concentration of MSAL-type alkaloids, including methyllycaconitine (MLA). However, the norditerpenoid alkaloids found in the greatest concentrations in most D. barbeyi populations are either deltaline or 14-O-acetyldictyocarpine (14-OAD), both less toxic MDL-type alkaloids. Although the individual toxicities of MLA, 14-OAD, and deltaline have been determined, the impact (additive or antagonistic) that large concentrations of deltaline or 14-OAD in the plant have on the toxicity of MLA is unknown. Consequently, the effect of MDL-type alkaloids on the toxicity of MLA was compared by using median lethal dose (LD(50)) and toxicokinetic profiles of the brainand muscle from mice receiving i.v. administration of these alkaloids, individually or in combination, at ratios of 1:1, 1:5, and 1:25 MLA to MDL-type alkaloids. The LD(50) for MLA alone was 4.4 +/- 0.7 mg/kg of BW, whereas the coadministration of MLA and deltaline at 1:1, 1:5, and 1:25 resulted in an LD(50) of 2.7, 2.5, and 1.9 mg/kg of BW, respectively. Similarly, the coadministration of MLA and 14-OAD at 1:1, 1:5, and 1:25 resulted in an LD(50) of 3.1, 2.2, and 1.5 mg/kg of BW, respectively. Coadministration of mixtures did not result in increased MLA bioavailability or alterations in clearance from the brain and muscle. Consequently, the increased toxicity of the mixtures was not a result of increased MLA bioavailability (based on the maximum concentrations observed) or alterations in MLA clearance from the brain and muscle, because these were unchanged. These results demonstrate that MDL-type alkaloids have an additive effect on MLA toxicity in mice and may also play a role in the overall toxicity of tall larkspur plants in cattle.

Coniine, one of the major toxic alkaloids present in poison hemlock ( Conium maculatum), occurs in two optically active forms. A comparison of the relative potencies of (+)- and (-)-coniine enantiomers has not been previously reported. In this study, we separated the enantiomers of coniine and determined the biological activity of each enantiomer in vitro and in vivo. The relative potencies of these enantiomers on TE-671 cells expressing human fetal nicotinic neuromuscular receptors had the rank order of (-)-coniine > (+/-)-coniine > (+)-coniine. A mouse bioassay was used to determine the relative lethalities of (-)-, (+/-)-, and (+)-coniine in vivo. The LD 50 values of the coniine enantiomers were 7.0, 7.7, and 12.1 mg/kg for the (-)-, (+/-)-, and (+)- forms of coniine, respectively. The results from this study demonstrate that there is a stereoselective difference in the in vitro potencies of the enantiomers of coniine that directly correlates with the relative toxicities of the enantiomers in vivo.

Introduction: Ammodendrine (1), anabasine (2) and coniine (3) can cause congenital malformations in livestock. They appear naturally in both enantiomeric forms, and can cause variable physiological responses. A method to measure the enantiomeric ratio of these natural toxins is needed., Objective: To develop a simple and economical method in order to determine the enantiomeric ratios of piperidine and pyrrolidine alkaloids in small samples of plant material., Methodology: Mixtures of isolated or purified plant alkaloids were converted to their Fmoc-L-Ala-alkaloid analogues forming diastereomeric mixtures, which were then analysed by high pressure liquid chromatography (HPLC) with mass spectrometry (MS) and ultraviolet (UV) detection to determine enantiomeric ratios., Results: The diastereomeric analogs for ammodendrine, anabasine and nornicotine could be separated and the enantiomeric ratios determined. The Fmoc-L-Ala-coniine analogue was not resolved under the HPLC conditions studied. The enantiomeric ratios of the selected plant alkaloids were measured and found to differ between both location within a species and location between species., Conclusion: A low-cost HPLC method to analyse the enantiomeric ratio of plant alkaloids containing primary or secondary amine nitrogens via conversion to their respective diastereomeric analogues has been developed.

Background: Failure of palatal shelf fusion at the precise gestational time point when shelf elevation and migration normally occur results in clefting. The present study defined the mechanism of clefting in the congenital caprine model by evaluating the temporal sequence of palatal shelf fusion., Methods: Six Spanish-type goats pregnant with twins were gavaged for 10 days (gestational days 32 to 41) with anabasine. Goats were examined with ultrasound throughout this period to assess fetal movement and evaluated histologically. Twelve untreated, unclefted fetuses served as controls., Results: After anabasine induction, real-time ultrasound confirmed a dramatic decrease in fetal movement. Hyperflexion of the neck occurred throughout the experimental period compared with control fetuses, in which spontaneous neck movements occurred. Histologic evaluation of the induced fetuses demonstrated the tongue wedged between the palatal shelves throughout the period of fusion. The shelves remained vertically oriented without elevation, ultimately resulting in clefting. The unclefted, control group demonstrated progressive elevation and migration of the palatal shelves between gestational days 38 and 40, with contact and fusion of the medial edge epithelia in the midline., Conclusions: Palatal clefting in the congenital caprine model occurred concomitant with reduced fetal movement. Resultant neck hyperflexion causes the tongue to obstruct medial migration of the palatal shelves and fusion, whereas the shelves themselves are either directly prevented from elevation secondary to anabasine impairment or indirectly secondary to the obstructing tongue. Although the molecular mechanism for the teratogenic effects of anabasine remains elusive, the authors have demonstrated an association between reduced fetal movement and palatal clefting in their congenital caprine model.

The aim of the present study was to determine and compare the clinical, hematological, biochemical and histopathological changes induced by cyanide, thiocyanate and chokecherry (Prunus virginiana) in goats. Sixteen Boer-Spanish cross-bred female goats were divided into four treatment groups: (1) control, (2) potassium cyanide (KCN) at 3.8 mg kg(-1) day(-1), (3) potassium thiocyanate (KSCN) at 4.5 mg kg(-1) day(-1) and (4) ground frozen chokecherry leaves and flowers at a target dose of 2.5 mg HCN kg(-1) day(-1), all for 4 weeks. Clinical signs were observed in two goats treated with chokecherry. Only sporadic changes were found in the hematological and blood chemical panel. Goats treated with chokecherry and thiocyanate had an increased number of vacuoles in the colloid of thyroid glands. Spongiosis and spheroids were found in the mesencephalon from goats treated with KCN and chokecherry. These findings suggest the thyroid lesions can be attributed to thiocyanate, whereas the effects on the nervous system were most likely caused by cyanide.

Objective: Despite cleft palate repair, velopharyngeal competence is not achieved in approximately 15% of patients, often necessitating secondary surgical correction. Velopharyngeal competence postrepair may require the conversion of levator veli palatini muscle fibers from injury-susceptible type 2 fibers to injury-resistant type 1 fibers. As an initial step to determining the validity of this theory, we tested the hypothesis that, in most cases, repair induces the transformation to type 1 fibers, thus diminishing susceptibility to injury., Interventions: Single permeabilized levator veli palatini muscle fibers were obtained from normal palates and nonrepaired congenitally-clefted palates of young (2 months old) and adult (14 to 15 months old) goats and from repaired palates of adult goats (8 months old). Repair was done at 2 months of age using a modified von Langenbeck technique., Main Outcome Measures: Fiber type was determined by contractile properties and susceptibility to injury was assessed by force deficit, the decrease in maximum force following a lengthening contraction protocol expressed as a percentage of initial force., Results: For normal palates and cleft palates of young goats, the majority of the fibers were type 2 with force deficits of approximately 40%. Following repair, 80% of the fibers were type 1 with force deficits of 20% +/- 2%; these deficits were 45% of those for nonrepaired cleft palates of adult goats (p < .0001)., Conclusion: The decrease in the percentage of type 2 fibers and susceptibility to injury may be important for the development of a functional levator veli palatini muscle postrepair.

Inherent differences in the levator veli palatini (LVP) muscle of cleft palates before palatoplasty may play a role in persistent postrepair velopharyngeal insufficiency (VPI). Contractile properties of LVP muscle fibers were analyzed from young (2-month) normal (YNP), young congenitally cleft (YCP) and again on the same YCP subjects 6 months after palatoplasty, mature repaired palate (MRP). The cross-sectional area and rate of force development (ktr) were measured. Specific force (sF(0)) and normalized power (nP(max)) were calculated. Using k(tr) to determine fiber type composition, YNP was 44% type 1 and 56% type 2, while YCP was 100% type 2. Two MRP subjects shifted to 100% type 1; 1 demonstrated increased resistance to fatigue. No differences in sF(0) were observed. nP(max) increased with presence of type 2 fibers. The persistent state of type 2 fibers following palatoplasty leads to increased fatigue in the LVP of MRP subjects and may cause VPI symptoms.

Several lupines (Lupinus spp.) present on western U.S. rangelands contain alkaloids that are teratogenic to livestock and cause congenital birth defects in calves (crooked calf disease). Periodically, large losses of calves due to lupine-induced "crooked calf disease" occur in northern Oregon and eastern Washington state. Five lupine populations from this area representing three species (L. leucophyllus, L. sulfureus, and L. sericeus) were evaluated taxonomically and by gas chromatography/mass spectrometry, and the major alkaloids in each lupine species were identified. The teratogenic alkaloid anagyrine was present in both of the lupine species responsible for the high outbreaks in east-central Washington and northeastern Oregon. However, the alkaloid profiles of the two lupines identified as L. leucophyllus were dissimilar, as were the alkaloid profiles of the two lupines identified as L. sulfureus. Botanical classification is not sufficient to determine potential teratogenicity, and it must be followed by chemical characterization to determine risk to livestock.

Phytochemicals are constitutive metabolites that enable plants to overcome temporary or continuous threats integral to their environment, while also controlling essential functions of growth and reproduction. All of these roles are generally advantageous to the producing organisms but the inherent biological activity of such constituents often causes dramatic adverse consequences in other organisms that may be exposed to them. Nevertheless, such effects may be the essential indicator of desirable properties, such as therapeutic potential, especially when the mechanism of bioactivity can be delineated. Careful observation of cause and effect, followed by a coordinated approach to identify the responsible entities, has proved extremely fruitful in discovering roles for phytochemical constituents. The process is illustrated by selected examples of plants poisonous to animals and include the steroidal alkaloid toxin of Veratrum californicum (Western false hellebore), piperidine alkaloids of Lupinus species (lupines), and polyhydroxy indolizidine, pyrrolizidine and nortropane alkaloids of Astragalus and Oxytropis species (locoweeds), Castanospermum australe (Moreton Bay chestnut) and Ipomoea species (morning glories).

Craniofacial anomalies, eye malformations, and permanent flexures of the forelimbs are common malformations seen in ruminants grazing semiarid rangelands of Northeastern Brazil. To investigate the cause of these malformations, we fed 2 suspected plants, Mimosa tenuiflora or Prosopis juliflora, to groups of 4 pregnant goats each. Fresh green M. tenuiflora was collected daily and fed ad libitum to 4 goats in group 1 throughout pregnancy. This treatment group also received a supplemental feed concentrate equivalent to 1% body weight. Four goats in group 2 received a ration with 70% of P. juliflora pods and 30% hay throughout pregnancy. Four control goats were fed supplemental feed concentrate (1% body weight) and hay ad libitum throughout pregnancy. Goats treated with P. juliflora pods and the control goats delivered 9 normal kids. The four goats that were fed M. tenuiflora during pregnancy delivered 4 kids, 3 of which had abnormalities similar to those observed in field cases, including cleft lip, unilateral corneal opacity, ocular bilateral dermoids, buphthalmos with a cloudy brownish appearance of the anterior chamber due to an iridal cyst, and segmental stenosis of the colon. Malformations induced experimentally by M. tenuiflora were similar to those observed in field cases, suggesting that M. tenuiflora is a cause of the field cases observed in the Brazilian semiarid rangelands.

Microvascular corrosion casts of caruncles from non-pregnant and pregnant doe goats at 4, 7, 10, 13, 16, and 18 weeks were examined with scanning electron microscopy. The internal convex surface of the caruncles of non-pregnant does was covered with capillary meshes of regular diameter and form, without crypts. As pregnancy advanced the complexity of the vasculature increased: at 4 weeks the surface showed a pattern of ridges separated by troughs. At later stages, branches of radial arteries penetrated the periphery forming an extensive mesh of capillaries on the concave surface. Capillary diameters increased significantly during pregnancy, especially after 4 weeks, when large flattened sinusoids formed. These sinusoids had a great deal of surface area for potential contact with the fetal component. The caprine placenta is usually considered to have increased interhemal distance compared with endotheliochorial and hemochorial types: our results suggest that the very extensive development of sinusoids and crypts may compensate for any negative consequences of the placental architecture. Placental angiogenesis, which is physiologically normal, may serve as a general model of this process in other circumstances, such as tumor. The effect of swainsonine (active compound of locoweed and a potential anticancer drug) on vascular development showed no differences in sinusoidal diameters at 7 weeks, but a decrease in capillary density was noted. Swainsonine caused a great distortion to the vasculature at 18 weeks. The effects of this compound on the vascular development lend credibility to its potential as an anticancer agent., ((c) 2007 Wiley-Liss, Inc.)

Background: Analysis of the composition of muscle fibers constituent to a cleft palate could provide significant insight into the cause of velopharyngeal inadequacy. The authors hypothesized that levator veli palatini muscle dysfunction inherent to cleft palates could affect the timing and outcome of cleft palate repair., Methods: Single, permeabilized muscle fibers from levator veli palatini muscles of three normal (n = 19 fibers) and three chemically induced congenital cleft palates (n = 21 fibers) of 14-month-old goats were isolated, and contractile properties were evaluated. The maximum isometric force and rate constants of tension redevelopment (ktr) were measured, and the specific force and normalized power were calculated for each fiber., Results: The ktr measures indicate that cleft fibers are predominantly fast-fatigable; normal fibers are slow fatigue-resistant: after a 10-minute isometric contraction, fibers from cleft palates had a loss of force 16 percent greater than that from normal palates (p = 0.0001). The cross-sectional areas of the fibers from cleft palates (2750 +/- 209 microm2) were greater (p = 0.05) than those from normal palates (2226 +/- 143 microm2). Specific forces did not differ between the two groups. Maximum normalized power of fibers from cleft palates (11.05 +/- 1.82 W/l) was greater (p = 0.0001) than fibers from normal palates (1.60 +/- 0.12 W/l)., Conclusions: There are clear physiologic differences in single muscle fibers from cleft palates and normal palates: cleft palate fibers are physiologically fast, have greater fatigability, and have greater power production. Detection of functional and/or fiber type differences in muscles of cleft palates may provide preoperative identification of a patient's susceptibility to velopharyngeal inadequacy and permit early surgical intervention to correct this clinical condition.

Objective: Levator veli palatini muscles from normal palates of adult humans and goats are predominantly slow oxidative (type 1) fibers. However, 85% of levator veli palatini fibers from cleft palates of adult goats are physiologically fast (type 2). This fiber composition difference between cleft and normal palates may have implications in palatal function. For limb muscles, type 2 muscle fibers are more susceptible to lengthening contraction-induced injury than are type 1 fibers. We tested the hypothesis that, compared with single permeabilized levator veli palatini muscle fibers from normal palates of adult goats, those from cleft palates are more susceptible to lengthening contraction-induced injury., Interventions: Congenital cleft palates were the result of chemically-induced decreased movement of the fetal head and tongue causing obstruction of palatal closure. Each muscle fiber was maximally activated and lengthened., Outcome Measures: Fiber type was determined by contractile properties and gel electrophoresis. Susceptibility to injury was assessed by measuring the decrease in maximum force following the lengthening contraction, expressed as a percentage of the initial force., Results: Compared with fibers from normal palates that were all type 1 and had force deficits of 23 +/- 1%, fibers from cleft palates were all type 2 and sustained twofold greater deficits, 40 +/- 1% (p = .001)., Conclusion: Levator veli palatini muscles from cleft palates of goats contain predominantly type 2 fibers that are highly susceptible to lengthening contraction-induced injury. This finding may have implications regarding palatal function and the incidence of velopharyngeal incompetence.

Lupinus leucophyllus is one of many lupine species known to contain toxic and/or teratogenic alkaloids that can cause congenital birth defects. The concentrations of total alkaloids and the individual major alkaloids were measured in three different years from different plant parts over the phenological development of the plant. All of the alkaloids were found in the different plant tissues throughout the growing season, although their levels varied in different tissues. Concentrations of total alkaloids and the individual alkaloids varied on an annual basis and in their distribution in the different tissues. Anagyrine levels were highest in the floral tissue, lupanine and unknown F accumulated to the greatest level in the vegetative tissue, and 5,6-dehydrolupanine accumulated to the highest level in the stem. These alkaloids appear to be in a metabolically active state with the teratogenic alkaloid anagyrine accumulating to its highest level in the developing seed. The latter is, thus, the phenological stage posing the greatest danger to grazing livestock.

Background: Midfacial hypoplasia and growth disturbances following cleft palate repair are well-known consequences of the maxillary scarring inherent with each repair technique. The present study investigated the influence of in utero palatoplasty, and cleft repair in 6-week-old goats, on midfacial growth and bony palatal development in the authors' congenital caprine cleft palate model in an effort to identify an inherent component of facial growth impairment associated with the cleft anomaly., Methods: At 85 days' gestation, eight clefted fetuses underwent in utero cleft palate repair using a modified von Langenbeck technique; eight fetuses remained as unrepaired controls. At 6 weeks of age, eight goats underwent cleft palate repair using the same technique. All goats were euthanized at 6 months of age; dry skull measurements and cephalometric analyses were performed., Results: Fetal Repairs: Both repaired and unrepaired clefted goats demonstrated significant evidence of maxillary hypoplasia compared with unclefted controls. Repaired goats showed a decrease of 15.7 percent in maxillary length, and unrepaired clefted goats showed a decrease of 18.0 percent in maxillary length, compared with unclefted controls. There was no significant difference in maxillary growth between the repaired and unrepaired clefted groups. Bony cleft width was reduced by 48 percent anteriorly and 60 percent posteriorly. Thirty-seven percent of repaired congenital clefts demonstrated partial bony fusion involving 10 to 70 percent of the palatal length. This was accompanied by an 8.8 percent decrease in maxillary width at the level of the third molar crown compared with unclefted controls and an 18.3 percent decrease in maxillary width compared with unrepaired clefted goats. Unrepaired clefted goats demonstrated neither a decrease in maxillary width nor any narrowing or fusion of the bony cleft. Newborn Repairs: Significant midfacial growth impairment was seen in animals that underwent cleft palate repair at 6 weeks of age compared with those repaired in utero and with unclefted controls. Repaired clefted goats demonstrated a significant decrease in maxillary length by 29.5 percent compared with unclefted controls and 16.4 percent compared with the group repaired in utero. Repaired goats also demonstrated a significant decrease in maxillary width in the transverse dimension at the level of the third molar. A decrease in maxillary width of 25.3 percent was seen compared with the unclefted controls and 18.1 percent compared with the fetal repairs. Bony cleft width was reduced by 32 percent anteriorly and 27 percent posteriorly following repair at 6 weeks of age. Although all goats demonstrated narrowing following repair, partial bony palatal fusion was not seen in this group., Conclusions: In utero cleft palate repair does not contribute to impairment of midfacial growth. The authors attribute this finding to the scarless nature of mucoperiosteal healing in the privileged fetal environment. However, the cleft palate anomaly does have an inherent component of facial dysmorphology that is evidenced as maxillary hypoplasia or retrusion in unrepaired clefted animals. Cleft repair in the newborn period, or thereafter, results in midfacial growth impairment in a manner similar to that seen clinically. The authors attribute this finding to the scarring that routinely accompanies conventional palatoplasty. The combination of the growth impairment inherent in the cleft anomaly and that attributable to postnatal repair scarring yields the midfacial retrusion commonly associated with cleft palate.

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.

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.

Objective: To evaluate the respiratory excretion and elimination kinetics of organic and inorganic selenium after oral administration in sheep., Animals: 38 crossbred sheep., Procedures: Selenium was administered PO to sheep as a single dose of 0, 1, 2, 3, or 4 mg/kg as sodium selenite or selenomethionine. Expired air was collected and analyzed from all sheep at 4, 8, and 16 hours after administration., Results: Clinical signs consistent with selenium intoxication were seen in treatment groups given sodium selenite but not in treatment groups given the equivalent amount of selenium as selenomethionine. However, a distinct garlic-like odor was evident in the breath of all sheep receiving 2 to 4 mg of selenium/kg. The intensity of odor in the breath did not correlate with clinical signs in affected animals receiving sodium selenite treatment., Conclusions and Clinical Relevance: The concentration of selenium in expired air was greater in sheep receiving selenium as selenomethionine than sodium selenite. The concentration of selenium in expired air from sheep receiving high doses of selenium (3 and 4 mg of selenium/kg) was larger and selenium was expired for a longer duration than the concentration of selenium in expired air from sheep receiving low doses of selenium (1 and 2 mg of selenium/kg).

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.

alpha-Solanine and alpha-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 6 microM of alpha-solanine, alpha-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 6 microM of alpha-solanine, alpha-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.

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.

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.

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).

Isocupressic acid (ICA) [15-hydroxylabda-8 (17), 13E-dien-19-oic acid], a labdane diterpene acid, isolated from ponderosa pine (Pinus ponderosa), Lodgepole pine (Pinus contorta), common juniper (Juniperus communis) and Monterey cypress (Cupressus macrocarpa), induces abortion in pregnant cows when ingested primarily during the last trimester. The objective of this study was to investigate the effects of isocupressic acid on bovine oocyte maturation (in vitro maturation (IVM)-Experiment I) and preimplantation embryo development (in vitro culture (IVC)-Experiment II) using in vitro embryo production techniques and to subsequently evaluate viability and developmental competence of ICA-cultured embryos via embryo transfer to recipient heifers (Experiment III). A complete randomized block experimental design was used. In Experiment I and II, isocupressic acid was added to IVM or IVC media at 0 (TRT1, control), 1.3 (TRT2), and 2.6 microg/ml (TRT3) Results from Experiment I and II indicated that ICA did not inhibit oocyte maturation and did not adversely affect preinpiantation embryo development. Furthermore, results from Experiment II demonstrated that isocupressic acid enhanced bovine preimplantation embryo development in vitro in a dose dependent manner. Subsequently, Day 8 (Day 0 = IVF) blastocysts cultured in vitro in the medium containing 2.6 microg/ml ICA were transferred to recipient heifers and resulted in normal pregnancies as determined by ultrasound imaging. Subsequently, all but two births were normal as evaluated by post natal veterinary examination. In conclusion, ICA showed no adverse effects on oocyte maturation and preimplantation embryo development in vitro or subsequent viability in vivo using the ICA concentrations and in vitro culture parameters of this study.

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.

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.

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 role of fetal surgery in the management of congenital anomalies and intrauterine abnormalities is appropriately restricted on the basis of feasibility and risk-to-benefit analyses of intrauterine intervention. Recently, the authors demonstrated that in utero cleft palate repair of the congenital caprine model is technically feasible and results in scarless healing of the mucoperiosteum and velum, with subsequent development of a potentially functional bilaminar palate with distinct oral and nasal mucosal layers, following single-layer repair of the fetal mucoperiosteal flaps. A slight indentation at the site of repair was the only remaining evidence of a cleft. At 6 months of age, normal palatal architecture, including that of mucosal, muscular, and glandular elements, was seen grossly and histologically. The present work investigated the ultrastructural and functional aspects of the palate following in utero cleft repair to determine what benefits might be derived from fetal intervention. Six goats pregnant with twins were gavaged twice daily for 10 days (gestational days 32 to 41; term, 145 days) with dry, ground Nicotiana glauca plant delivering between 2.4 and 14 mg/kg per day of anabasine, doses that were adjusted in response to mater-nal toxicity. At 85 days' gestation, six fetuses underwent in utero palatoplasty using a modified von Langenbeck technique with elevation of bilateral mucoperiosteal flaps and lateral relaxing incisions. A single-layer repair of the mucoperiosteal flaps was performed using interrupted 6-0 Vicryl sutures. Six fetuses remained as unrepaired clefted controls. Six months after in utero palatoplasty, each group of goats underwent nasoendoscopy to evaluate palatal function; two unclefted 6-month-old goats served as controls. Subsequently, soft palate muscle was harvested from each of the goats and was evaluated by light and electron microscopy. Velar muscle was also harvested from the unclefted control goats and was similarly studied. Nasoendoscopy demonstrated functional palates capable of dynamic velopharyngeal closure following in utero cleft repair; this motion was similar to that observed in unclefted animals. Unrepaired clefted goats did not demonstrate any evidence of velar motion or velopharyngeal closure. Soft palate muscle from this group demonstrated evidence of myofibril degeneration, atrophy, and loss compared with unclefted control velar muscle. Ultrastructural changes included sarcomere "scalloping, " partial Z-line degeneration and loss, and progressive I-band degeneration and loss. Repaired clefted soft palate muscle was remarkably similar to unclefted control muscle. Significantly less myofibril, Z-line, and I-band degeneration and loss were observed with minimal evidence of sarcomere scalloping. In utero cleft palate repair results in a functional soft palate with restoration of ultrastructural architecture of the velum. These findings were attributed to reconstitution of the velar muscular sling, which is disrupted during the clefting process and remains abnormally inserted into the posterior edge of the palatal bone and along the bony cleft. Although repaired velar muscle does demonstrate some evidence of ultrastructural change compared with control muscle, these findings are significantly less pronounced than those observed in the unrepaired clefted muscle.

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.

Ten yearling steers weighing 270-360 kg died acutely after eating early seed pod stage Lupinus argenteus (silvery lupine) containing high levels of piperidine alkaloids, including ammodendrine and N-methylammodendrine. Reduced availability of Quality feed from range moderately depleted of Quality grasses appeared a major factor in causing the cattle to graze lupine. Proximate analysis of whole plant, seed pods and seed showed high levels of crude protein ranging from 16.4 to 48% for whole plant and seed. Alkaloid analysis of the whole plant and individual plant parts such as seed and seed pods (30-40% seed shatter) showed total alkaloid levels ranging from 0.70 to 2.5% (0.70-2.5 mg/100 mg plant) dry weight. Ammodendrine levels ranged from 0.24% in seed pods with >70% seed loss to 1.35% in early growth plant with no seed. N-methylammodendrine levels ranged from 0.38% in seed pods with >70 % seed shatter to 1.04 % in pure seed. Other minor alkaloids totaled 0.08% in seed pods with >70% seed shatter to 0.60% in early growth plant before seed pod formation. These cattle ate an estimated 1.8 g/kg dry weight (486 g to 648 g) of lupine in 24 h. This appears the first report of acute death in cattle associated with piperidine alkaloid-containing Lupinus argenteus.

The effects of two commonly used cell culture mitogens, pokeweed (PWM) and phytohemagglutinin (PHA) on bovine oocyte maturation in vitro (IVM) and preimplantation embryo development in vitro were evaluated by randomized complete block experimental design with three treatments. Effects were measured by quantifying subsequent embryo development. Oocyte maturation was adversely affected by PWM-containing medium as indicated by a decrease in cleavage rate and subsequent embryo development to morula and blastocyst stages. Embryo developmental competence was also adversely affected by PWM. Development in PHA-containing medium was significantly better (P<0.05) than in the PWM treatment, although there was no difference (P>0.05) when compared to Control. We conclude that there are no beneficial effects in adding mitogenic agents to culture medium to enhance in vitro embryo production and development.

Reproductive dysfunction has many potential causes. In this article, the authors discuss toxicants that have been shown to affect reproduction, with emphasis on food-producing animals and fowl. The discussion is brief and written to provide a resource for clinicians, students, and scientists by focusing on toxicant-induced reproductive dysfunction and discussing the toxicant, source, clinical effects, and livestock species known or suspected to be affected.

Three new N-(methylsuccinimido)anthranoyllycoctonine norditerpenoids, given the names bearline (1), 14-acetylbearline (2), and 16-deacetylgeyerline (3), were isolated from the extract of the low larkspur Delphinium nuttallianum. The structures of the individual alkaloids were determined by (1)H and (13)C NMR spectroscopy and HRMS. These alkaloids are structurally related to the neurotoxic alkaloid methyllycaconitine (4) and may be important in establishing the toxicity of low larkspurs to cattle. A mouse bioassay was used to measure the LD(50) values for two of the new alkaloids (1 and 2), as sufficient quantities of 3 were not available for toxicity testing. A structurally related alkaloid, geyerline (7), was isolated from D. geyeri in sufficient quantities for toxicity testing. The toxicities of 1, 2, and 7 were found to be comparable to that of 4, with calculated LD(50) values in mice of 5.7, 3.3, and 6.2 mg/kg, respectively.

The induction of cleft palate by Nicotiana glauca (wild tree tobacco) during the first trimester of pregnancy was compared between Spanish-type goats and crossbred western-type sheep. Cleft palate was induced in 100% of the embryonic/fetal goats when their pregnant mothers were gavaged with N. glauca plant material or with anabasine-rich extracts from the latter, during gestation days 32-41. Seventy-five percent of newborn goats had cleft palate after maternal dosing with N. glauca during gestation days 35-41, while no cleft palates were induced when dosing periods included days 36-40, 37-39, or day 38 only. The induced cleft palates were bilateral, involving the entire secondary palates with complete detachment of the vomer. Eleven percent of the newborn goats from does gavaged during gestation days 32-41 had extracranial abnormalities, most often contractures of the metacarpal joints. Most of these contractures resolved spontaneously by 4-6 weeks postpartum. One newborn kid also had an asymmetric skull due to apparent fetal positioning. No cleft palates were induced in lambs whose mothers were gavaged with N. glauca plant or anabasine-rich extracts during gestation days 34-41, 35-40, 35-41, 36-41, 35-51, or 37-50. Only one of five lambs born to three ewes gavaged with N. glauca plant material during gestation days 34-55 had a cleft palate, but all five of these lambs had moderate to severe contractures in the metacarpal joints. The slight to moderate contracture defects resolved spontaneously by 4-6 weeks postpartum, but the severe contractures resolved only partially. Embryonic/fetal death and resorption (determined by ultrasound) occurred in 25% of pregnant goats fed N. glauca compared to only 4% of pregnant sheep. Nicotiana glauca plant material contained the teratogenic alkaloid anabasine at 0.175% to 0.23%, dry weight, demonstrating that Spanish-type goats are susceptible to cleft palate induction by the natural toxin anabasine, while crossbred western-type sheep are resistant. However, clinical signs of toxicity were equally severe in goats and sheep, even though maternal alkaloid tolerance was generally lower in sheep. We postulate that an alkaloid-induced reduction in fetal movement during the period of normal palate closure is the cause of the cleft palate and multiple flexion contractures. Teratology 61:203-210, 2000. Published 2000 Wiley-Liss, Inc.

A rapid electrospray mass spectrometry method was developed for screening larkspur (Delphinium spp.) plant material for toxic norditerpenoid alkaloids. The method was calibrated using two standard alkaloids, methyllycaconitine (1) and deltaline (2), with a recovery of 92% from spiked samples and relative standard deviations of 6.0% and 8.1% for the two alkaloids, respectively. Thirty-three samples of plains larkspur, Delphinium geyeri, were analyzed. Methyllycaconitine (1) concentration was 0.27% +/- 0.08% during a 1-month period in 1997 establishing the relative risk of poisoning from the plant to be low. The method was also applied to the trace analysis (<1 ppm) of 1 in serum samples from sheep dosed different levels of the alkaloid. Electrospray ionization combined with sequential tandem mass spectrometry and HPLC coupled to atmospheric pressure chemical ionization (APCI) mass spectrometry were used to detect and tentatively identify three new norditerpenoid alkaloids from Delphinium nuttallianum [bearline (6), 14-acetylbearline (7), 16-deacetylgeyerline (8)]. The tentative structure of the new alkaloids was predicted from the tandem mass spectra fragmentation patterns and assigning the substitution pattern for methoxy and acetyl groups at the C-14 and C-16 carbons.

Locoweed (Oxytropis sericea) was fed to 4 open cycling cows that had repeatedly consumed locoweed in previous grazing trails. They received locoweed at 20% of their diet for 30 d (0.68-0.76 mg swainsonine/kg/d). Locoweed induced an immediate rise in serum swainsonine (the locoweed toxin) and a concomitant drop in serum alpha-mannosidase activity in all cows accompanied by abnormal estrus behavior, increased estrous cycle lengths and failure to conceive. Serum progesterone (P4) profiles demonstrated that estrous cycles lengthened from an average of 19 d before locoweed feeding to an average of 34 d (range 24-43 d) while on locoweed. After locoweed feeding ceased, normal estrous cycles returned within an average of 14 d (range 7-25 d). Two of the 4 cows conceived on their first post-locoweed estrus at 7 and 25 d. The third cow bred twice at 13 and 31 d after lowoweed feeding stopped, and the fourth cow bred 3 times at 11, 31 and 52 d before conception occurred. Pregnancies in all 4 cows progressed normally to 7 mo gestation when locoweed was again fed at 20% of the diet for 40 d (gestation days 213 and 253) to 2/4 cows, 1 of which aborted 10 d after lowoweed feeding stopped (263 days gestation). The other cow fed lowoweed calved normally as did the 2 pregnancy control cows. Serum P4 and estradiol (E2) profiles during pregnancy appeared normal before, during and after locoweed feeding except in the cow that aborted, whose P4 declined and E2 increased prematurely. The general trend of serum prolactin was similar in locoweed-fed and control cows.