Location: Forage-animal Production Research
2020 Annual Report
Objectives
Objective 1: Determine relationships of ergot alkaloids to receptors in animal tissues and subsequent effects on animal physiology, and the implications of these relationships on clearance from animal tissues.
Subobjective 1.A. Evaluate the effect of ergot alkaloid exposure on vascular biogenic amine receptors.
Subobjective 1.B. Characterize the interaction of isoflavones and their metabolites with ergot alkaloids on vasoactivity.
Subobjective 1.C. Determine if there is ergovaline in bovine portal blood and determine site of absorption.
Subobjective 1.D. Develop an oral endotoxin challenge model to determine the effects of fescue-derived alkaloid consumption on intestinal barrier function and innate immunity in cattle.
Objective 2: Develop cost-effective management approaches to alleviate or mitigate the adverse effects of fescue toxicosis on animal physiology and well-being.
Subobjective 2.A. Determine the impact of combining feeding soybean hulls (SBH) and red clover on weight gain performance of steers grazing toxic E+ tall fescue and mitigation of fescue toxicosis.
Subobjective 2.B. Compare vasorelaxation between different sources of isoflavones for goats exhibiting ergot alkaloid-induced vasoconstriction.
Subobjective 2.C. Determine if rumen tryptophan-utilizing bacteria will degrade ergot alkaloids in vivo.
Subobjective 2.D. Assess phenotypic variation of cattle in their susceptibility to fescue toxicosis.
Subobjective 2.E. Evaluate management approaches to cost effectively add value to cull cows and enhance ground beef quality.
Subobjective 2.F. Comparison of supplemental selenium form to ameliorate physiological and gene expression stress response parameters in white blood cell (WBC), pituitary, adrenal, kidney, and liver of growing steers consuming E+ or E- tall fescue seed.
Subobjective 2.G. Assess effects of animal temperament on fescue toxicosis-induced changes in animal growth and immunological responses.
Objective 3: Determine the biological mechanisms used by certain plant secondary metabolites to function as antimicrobials in ruminants and non-ruminants, and assess their impact on animal health, performance, and well-being.
Subobjective 3.A. Elucidate the antimicrobial mechanism of action of the red clover isoflavone, biochanin A, and determine antagonistic, additive or synergistic activity with other antimicrobials.
Subobjective 3.B. Determine the effect of biochanin A on the rates of antibiotic resistance in ruminants.
Subobjective 3.C. Determine the effect of clover phenolic compounds on nitrogen efficiency and weight gain in lambs.
Subobjective 3.D. Evaluate spent brewer’s yeast as a carrier for hops secondary metabolites (prenylated phloroglucinols) in ruminant production.
Approach
Experiments will be conducted to determine mechanisms by which ergot alkaloids interact with receptors in animal tissues and affect their physiology. Saphenous veins collected from cattle at a local abattoir will be used to determine in vitro if inhibition of the phospholipase C and protein kinase C enzymes will alleviate ergot alkaloid induced vasoconstriction of smooth muscle. An in vitro experiment will ascertain if plant secondary metabolites, isoflavones, can mitigate the vasoactivity caused by ergot alkaloids. Endothelium cells from saphenous cells will also be exposed to ergot alkaloids to assess their effects on receptor signaling by ß-arrestin and G proteins. Catheters will be inserted in in the hepatic, portal and mesenteric veins of six rumen-fistulated steers to determine if rumen infused ergovaline is absorbed by the rumen and small intestines, or if the ergopeptine is degraded by rumen microbes. Field experiments will be conducted to evaluate management approaches to mitigate fescue toxicosis. Rumens will be infused with ergot alkaloids at a diet concentration of 0.8 ppm ergovaline and combined with either ground soybean meal, red clover, white clover, or a no isoflavone control. Cross sectional luminal areas of the right carotid artery of each goat will be measured by color Doppler ultrasonography. A grazing experiment with steers will evaluate the effects of feeding soybean hulls and overseeding toxic endophyte-infected tall fescue with red clover on animal weight gain and well-being. Four combinations of with and without the two treatments will be used as treatments to determine cost effectiveness of the treatments and mitigation of fescue toxicosis. Ear notches and phenotype data will be collected from multiple cow herds to detect polymorphisms of certain genes associated with fescue toxicosis and determine if these polymorphisms can be used to predict genetic tolerance to toxic ergot alkaloids. Three management approaches (soybean hulls, chemical seed head suppression, and red clover) to mitigate fescue toxicosis will be compared for adding body condition and weight to cull cows that graze toxic endophyte-infected tall fescue in either the spring or fall. To assess if selenium can ameliorate fescue toxicosis and if ergot alkaloids suppress immune response and alter gene expression in the liver, steers will be fed selenium depleted diets for 28 days and then switched to either inorganic selenium or an inorganic and organically bound selenium treatments for the remaining 98 days of the trial. The steers will also be fed either endophyte-infected or endophyte-free seed for the final 42 days. Jugular blood will be periodically collected and there will be staggered euthanasia of the steers for tissue collection. Effects of biochanin A on rate of antibiotic resistance will be determined by feeding rumen fistulated steers with either 0, 3 or 6 g biochanin A/day/steer and collecting rumen and fecal samples and using metagenomic DNA for quantitative PCR screening for antibiotic resistant genes.
Progress Report
Subobjective 1.A. - Evaluate the effect of ergot alkaloid exposure on vascular biogenic amine receptors. One final experiment remains in the study of how ergot alkaloids interact with serotonin receptors. All other experiments looking at the impact that ergot alkaloids have on signal transduction, receptor population, and intracellular recruitment of internalization signaling have been conducted. Cell line and consumables to conduct the HTR2A receptor sequestration and internalization experiments have been procured.
Subobjective 1.B. - Characterize the interaction of isoflavones and their metabolites with ergot alkaloids on vasoactivity. Ergot alkaloids cause vasoconstriction and isoflavones cause vasodilation. Myograph experiments evaluating the interaction of isoflavones and ergot alkaloids in bovine vasculature have been completed. Data are being analyzed.
Subobjective 1.C. - Determine if there is ergovaline in bovine portal blood and determine site of absorption. Two experiments have been completed following the development and validation of a blood-ergovaline extraction method. Data have been analyzed and abstracts have been prepared for presentation.
Subobjective 1.D. - Develop an oral endotoxin challenge model to determine the effects of fescue-derived alkaloid consumption on intestinal barrier function and innate immunity in cattle. All experiments have been completed. Two additional experiments to investigate the impact of bromocriptine (synthetic ergot alkaloid) on: 1) on mTOR signaling pathway (a genetic system associated with animal growth) in muscle and whole-body protein turnover in beef cattle, and 2) glucose and insulin metabolism in healthy and insulin dysregulated horses have been conducted and a manuscript has been submitted for publication.
Subobjective 2.A. - Determine the impact of combining feeding soybean hulls (SBH) and red clover on weight gain performance of steers grazing toxic E+ tall fescue and mitigation of fescue toxicosis. The experiment has been delayed due to the retirement of an ARS Scientist. A publication has been submitted detailing the impact of overseeding red clover on steers grazing endophyte-tall fescue pastures.
Subobjective 2.B. - Compare vasorelaxation between different sources of isoflavones for goats exhibiting ergot alkaloid-induced vasoconstriction. A manuscript detailing the effect of legume source isoflavone content on mitigating tall fescue toxicosis is currently in preparation. The results of the vaso-active measurements in the study have been presented three times in 2020 to both stakeholder and scientific audiences (see below).
Subobjective 2.C. - Determine if rumen tryptophan-utilizing bacteria will degrade ergot alkaloids in vivo. All experiments have been completed for this subobjective. Collected data have been analyzed and the resulting manuscripts are in preparation. The publication of these data are dependent on sequencing and identification of isolated tryptohan-utilizing bacteria.
Subobjective 2.D. - Assess phenotypic variation of cattle in their susceptibility to fescue toxicosis. Original phenotyping with hair coat score was determined to not be variable enough (the desirable outliers were not being observed). A remote sensing cattle herd management system was purchased and installed at university farms in Kentucky, Tennessee, and Georgia to provide a more in-depth phenotype on tall fescue. Due to changes in the defined phenotype, genotyping samples have not been collected.
Subobjective 2.E. - Evaluate management approaches to cost effectively add value to cull cows and enhance ground beef quality. The grazing study portion is ongoing.
Subobjective 2.F. - Comparison of supplemental selenium form to ameliorate physiological and gene expression stress response parameters in white blood cell (WBC), pituitary, adrenal, kidney, and liver of growing steers consuming E+ or E- tall fescue seed. The grazing-whole animal portion of the experiment was completed, all tissues were collected and stored, and whole-animal performance and clinical blood metabolites statistically analyzed. A manuscript is being prepared.
Subobjective 2.G. - Assess effects of animal temperament on fescue toxicosis-induced changes in animal growth and immunological responses. Associated confinement study has been completed and calves for year two of the grazing experiment have been selected and assessed for temperament.
Subobjective 3.A. - Elucidate the antimicrobial mechanism of action of the red clover isoflavone, biochanin A, and determine antagonistic, additive or synergistic activity with other antimicrobials. A variety of experiments have been conducted using the Streptococcus bovis model.
Subobjective 3.B. - Determine the effect of biochanin A on the rates of antibiotic resistance in ruminants. Samples have been collected and analyzed for the number of antibiotic resistant bacteria. Isolates of predominant antibiotic resistant bacteria have been sequenced and identified. Steers on a high grain diet carried a substantial sub-population of tetracycline-resistant bacteria in the rumen. Supplementation with biochanin A resulted in a significant reduction (approx. 99.9%) in the number of tetracycline-resistant bacteria.
Subobjective 3.C. - Determine the effect of clover phenolic compounds on nitrogen efficiency and weight gain in lambs. Isolates have been obtained for in vitro testing of biochanin A and other clover phenolic compounds. Two feeding trials with lambs have been conducted: 2) To examine nitrogen metabolism by lambs in vivo. 1) To examine the effects of biochanin A on growth and carcass characteristics.
Subobjective 3.D. - Evaluate spent brewer’s yeast as a carrier for hops secondary metabolites (prenylated phloroglucinols) in ruminant production. The project is delayed. Unit scientists wrote a proposal for a Small Business Innovation Research Phase II grant with cooperators. The grant will expand the subobjective by adding a treatment that is more relevant to the industry than unprocessed spent yeast.
Accomplishments
1. Ergot alkaloid toxins found in tall fescue, the predominate forage in the Southeastern U.S., alter eating patterns in cattle. A multi-study project evaluated the impact that consumption of ergot alkaloids has on gut motility. It was hypothesized that gut motility would decrease, causing an increase in gut fill and result in the hallmark symptom of decreased feed intake when livestock are exposed to ergot alkaloids. A program was developed by University of Kentucky and ARS researchers using MATLAB, a multi-paradigm numerical computing environment, to define a meal using feed bunks on loadcells to measure feed consumption. This information was combined with rumen motility data. Results from these experiments demonstrated that previously observed gut fill in cattle dietarily exposed to ergot alkaloids have decreased ruminal contractions, slower eating patterns, and altered water intake patterns. Combined, this leads to increased gut fill, decreased feed intake, and the overall decrease in productivity observed in ruminants consuming feed contaminated with ergot alkaloids. Understanding the cause of decreased feed intake in cattle exposed to ergot alkaloids will permit strategies to be developed that mitigate the associated loss of cattle gain and producer profits.
2. Common phytochemicals are antimicrobial to bovine and human gut bacteria. It is now recognized that the bacteria and other microorganisms in the gut influence many aspects of animal and human health. It is not always clear how minor dietary components, like phytochemicals, impact gut bacteria. ARS researchers at Lexington, Kentucky, and University of Kentucky researchers examined the antimicrobial properties of a panel of phytochemicals that included nicotine from tobacco, capsaicin from peppers, curcumin from the spice, turmeric. The phytochemicals were tested with closely related bacterial species, where one was from a cow and one from a human. All of the tested phytochemicals had antimicrobial activity to a number of bacteria. These results show how harmless and beneficial bacteria can be impacted by plants that we and our livestock consume. It also shows how plants and phytochemicals might be used to treat harmful bacteria like Clostridioides difficile (commonly called "C. diff"). This is important not only to scientists, but also to producers seeking out natural solutions to problems previously addressed with pharmaceutical options that are becoming less available.
3. Red clover, a common pasture legume, improves rumen fermentation and growth performance in grazing cattle. Weight gain is a primary driver of profit for most cattle producers. ARS scientists at Lexington, Kentucky, looked at supplementing red clover hay at two levels to grazing steers, and its impact on the microbial community and growth performance. Red clover hay supplementation improved rumen fermentation by inhibiting wasteful bacteria and promoting fiber utilization and short chain fatty acid production. Additionally, steers supplemented with red clover hay had greater growth performance than protein-matched controls. In all cases, red clover hay supplemented at 15% of the diet was more effective than the 30% level. These results provide evidence that lower levels of red clover supplementation may be optimal for maximizing microbial community function and growth performance in grazing steers. The impact of this research is that beef producers can improve cattle performance through moderate supplementation with clover hay.
4. Grazing red clover reverses fescue toxicosis in cattle. The pasture legume red clover contains isoflavones that can improve blood flow that is negatively impacted in animals suffering from fescue toxicosis. As such, the presence of red clover interseeded into toxic tall fescue pastures is a known tool to mitigate fescue toxicosis, but the direct impact on blood flow and performance has not been assessed. ARS scientists in Lexington, Kentucky, evaluated the impact of interseeding red clover on cattle grazing endophyte-infected tall fescue. Cattle grazing pastures with interseeded red clover had greater growth performance and exhibited increased vasodilation post-grazing. The impact of this research is that interseeding red clover in endophyte-infected tall fescue pastures can reverse some of the negative effects associated with fescue toxicosis. Notably, this is the first study that has demonstrated that simply grazing interseeded red clover is an effective pasture management strategy to elicit the vasoactive benefits of isoflavones.
Review Publications
Harlow, B.E., Flythe, M.D., Kagan, I., Goodman, J.P., Klotz, J.L., Aiken, G.E. 2020. Isoflavone supplementation, via red clover hay, alters the rumen microbial community and promotes weight gain of steers grazing mixed grass pastures. PLoS One. 15(3): e0229200. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0229200.
Egert-Mclean, A.M., Sama, M.P., Klotz, J.L., McLeod, K.R., Kristensen, N.B., Harmon, D.L. 2019. Automated system for characterizing short-term feeding behavior and real-time forestomach motility in cattle. Computers and Electronics in Agriculture. 167:105037. https://doi.org/10.1016/j.compag.2019.105037.
Lakes, J.E., Richards, C.I., Flythe, M.D. 2019. Inhibition of Bacteroidetes and Firmicutes by select phytochemicals. Anaerobe. 61:102145. https://doi.org/10.1016/j.anaerobe.2019.102145.
Flythe, M.D., Harlow, B.E. 2019. Effects of hops (Humulus lupulus L.) beta-acids on short chain fatty acid production from complex carbohydrates by rumen microbiota. Advances in Microbiology. 9(12):983-992. https://doi.org/10.4236/aim.2019.912063.
Ahn, G., Riccioni, K., Avila, S., Klotz, J.L., Harmon, D.L. 2019. Ruminal motility, reticulo-ruminal fill and eating patterns in steers exposed to ergovaline. Journal of Animal Science. 98(1):1-11. https://doi.org/10.1093/jas/skz374.
