Mindy Hubert

Document Type

Thesis - University Access Only

Award Date


Degree Name

Master of Science (MS)

Department / School

Animal Science


The toxicity of leafy spurge after in vitro fermentation with normal or modified cattle rumen digesta was evaluated. Ruminal digesta was obtained from ruminally-fistulated steers on an alfalfa/grass hay diet. Seventy-five ml of whole digesta was mixed with 300 ml of buffer solution and 26 g of ground ( 1 mm screen) leafy spurge or alfalfa (control), gassed with CO2 and incubated at 39 ° C. For one treatment, 0.5 mg of neomycin sulfate antibiotic was added to the mixture in order to alter the population of microbes. Fermentations were stopped at 15 min, 6 h and 1 2 h by freezing the mixtures quickly in thin ziploc bags. Fermentation mixtures were extracted with petroleum ether, and the ether was evaporated from extracts before they were tested for toxicity using the Brine Shrimp Lethality Assay. Three shrimp assays were conducted. In Assay 1, leafy spurge was more toxic to shrimp than alfalfa was (P< 0.01) and less toxic at 15 min of fermentation compared to 6 h (P<0.01) and 12 h (P<0.01). For Assay 2, adding neomycin sulfate to the mixture did not affect the toxicity (P = 0.65), and leafy spurge was less toxic at 1 5 min compared to 6 h of fermentation for normal digesta (P<0.01) and modified digesta (P<0.02). In Assay 3, leafy spurge was more toxic than alfalfa was (P<0.01) and the addition of a modifier did not alter the toxicity of leafy spurge compounds to brine shrimp. These results indicate that leafy spurge contains compounds that are toxic to shrimp, and these toxins become more toxic with 6 h and 1 2 h of interaction with rumen microbes. Neomycin sulfate as applied did not alter microbial populations such that toxicity was altered. Leafy spurge fermentation samples from Experiment 1 were tested for the concentration of ingenol, a diterpene moiety known to be cytotoxic. lngenol standard (purchased from Sigma; 1 mg of parent alcohol dissolved in 1 ml acetonitrile) was prepared as a series of dilutions and injected into the HPLC to produce a concentration standard curve. This curve was used to determine the concentration of ingenol in the 1 5 min and 6 h fermentation samples from Experiment 1. HPLC results indicated the presence of ingenol (parent alcohol form) in the hydrolyzed 15 min fermentation sample at an average concentration of 0.0024 mg ingenol per mg of sample injected (0.24%) and present in the hydrolyzed 6 h fermentation sample at an average concentration of 0.0011 mg ingenol per mg of sample injected (0.11 %). These values correlated to 0.0074% (15 min sample) and 0.0038% (6 h sample) ingenols in the original leafy spurge/digesta material. Another set of fermentation samples were not hydrolyzed to observe differences between the two fermentation times. HPLC results indicated ingenol esters in each fermentation sample with relatively similar retentiontimes (peak 1 = 31.533 vs. 31 .400 min; peak 2 = 32.533 vs. 32.367 min for the 15 min and 6 h fermentation samples, respectively). These data suggest that ingenol esters are present after 1 5 min and 6 h of fermentation with cattle rumen microbes, and that increased exposure to microbial fermentation may alter ingenols into other toxic and aversive phytochemicals. The response of cattle to a novel feed was evaluated when animals were dosed with ingenol, a compound found in leafy spurge, and ingenol 3- benzoate, a derivative of ingenol that has shown tumor-promoting activity (Vogg et al. 1999). Seven black Angus-cross heifers (average body weight= 271 kg) were separated into two groups: control (n = 3) and ingenol (n = 4) per treatment. Millet (Trial 1) or beet pulp pellets (Trial 2) were offered as a novel feed on day 1 of each trial. Following a moderate consumption of the novel feed (0.14-0.82 kg when offered 0.91 kg), the animals were orally dosed with 0.0148 mg/kg body weight of ingenol or ingenol-3-benzoate dissolved in an ethanol/water vehicle or ethanol/water alone (controls). The novel feed was offered again on day 2 of each trial. Consumption of the novel feed was the response variable. In Trial 1, the dose of ingenol caused an aversion in only one of four animals. There was no significant difference between ingenol and control groups (P = 0.41). One calf was given a second dose of ingenol on day 2 after it consumed a significantly higher amount of millet. This calf exhibited an aversion when offered millet on day 3. In Trial 2, one dose of ingenol 3-benzoate caused an aversion in three of four animals, and two of three control animals increased their intake of novel feed following dosage. There was no significant difference between the treated and control groups for change in novel feed intake (P=0.30).

Library of Congress Subject Headings

Leafy spurge
Plant toxins
Rumen -- Microbiology
Cattle -- Digestive organs



Number of Pages



South Dakota State University