Research Project: Optimizing the Biology of the Animal-Plant Interface for Improved Sustainability of Forage-Based Animal Enterprises

Location: Forage-animal Production Research

2021 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. Final experiment evaluating endocytosis of serotonin receptor 2A completed and data are being analyzed. This work has been delayed due to Covid-19 pandemic. Subobjective 1.B. - Characterize the interaction of isoflavones and their metabolites with ergot alkaloids on vasoactivity. The tissue culture work associated with this subobjective is partially complete and the cell culture work will be started later this year. Covid 19 restrictions have significantly delayed cell culture experiments from being conducted on time, but it is expected that they will be completed in the coming year. Subobjective 1.C. - Determine if there is ergovaline in bovine portal blood and determine site of absorption. Work has been completed and manuscript is in preparation. Challenges have arisen due to travel of the post-doc conducting the work being interrupted due to Covid-19 pandemic and the individual was not able to return to the laboratory to continue the research. 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. This work has been fully met and a manuscript is in preparation. 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 was delayed due to the retirement of an ARS scientist. A publication is currently under review at Applied Animal Science on overseeding red clover on mitigating fescue toxicosis in grazing steers. Subobjective 2.B. - Compare vasorelaxation between different sources of isoflavones for goats exhibiting ergot alkaloid-induced vasoconstriction. Manuscript detailing the effect of legume source isoflavone content on mitigating tall fescue toxicosis is currently in preparation. 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 tryptophan-utilizing bacteria. These analyses will not be available until after COVID restrictions are lifted at which time completion and submission of the manuscript will be done. 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 in sufficient quantity). A cow monitoring system was installed at university farms in Kentucky, Tennessee, and Georgia to provide more in-depth phenotype information on tall fescue pastures. Due to changes in the defined phenotype, genotyping samples have not been collected. A new phenotype is being developed using the results of this remote monitoring system. Subobjective 2.E. - Evaluate management approaches to cost effectively add value to cull cows and enhance ground beef quality. Study is ongoing. The red clover hay has been harvested and cull cows are being identified. Covid-19 pandemic has delayed procurement of cattle and labor. It expected that the work will be completed one year later than projected. 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 endophyte-infected or endophyte-free 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. Manuscripts are being prepared and bioinformatic analysis gene expression patters is ongoing. 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 the Year 2 grazing experiment has been completed and all data have been analyzed. Manuscripts are under preparation. 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, including dose-response curves with biochanin A and tetracycline delivered at different growth phases. Progress has been delayed by 1) FY2019 budget shutdown, 2) FY2020 pandemic response and 3) the retirement of the Biological Science Technician performing the experiments. The delay is expected to result in the work being completed one year later than projected. 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 SBIR Phase II 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 alkaloids lower serotonin concentrations in cattle. A multi-study project with ARS researchers and University of Kentucky and State University of Western Parana (Brazil) collaborators evaluated the impact that consumption of ergot alkaloids has on circulating levels of the neurotransmitter serotonin. Serotonin is a multifaceted neurotransmitter involved in cognitive, regulatory, and stimulatory functions that are also processes negatively affected by fescue toxicosis. A serotonin precursor was infused post-ruminally to verify that blood serotonin could be measured and altered. A follow-up series of studies in cattle were conducted that demonstrated that consuming ergot alkaloids in toxic tall fescue suppressed circulating serotonin levels. Suppression of serotonin was partially mitigated by inclusion of the serotonin precursor in the diet. Given the numerous physiologic roles serotonin plays throughout the body such as vascular tone and gut motility, the impact of understanding the relationship between ergot alkaloids and lowered circulating serotonin may be critical to the development of future mitigation strategies for fescue toxicosis.

2. Not all forage dietary fibers are equal when metabolized by the hindgut. Fructan and cellulose are both considered dietary fiber by nutritionists, but they are metabolized differently by gut microbiota. In a study with ARS researchers and University of Kentucky collaborators, fructan or cellulose were given to an uncultivated mixture of bacteria and other microorganisms that were harvested from mice. Analysis of the metabolic products from each fiber type revealed a great diversity of products made by the bacteria. In general, more compounds, including both fermentation end-products and anabolic products, were made from fructan than from cellulose. The impact of this research is a better understanding of dietary fiber to benefit human and animal health.

3. Biochanin A as an alternative to antibiotics for mitigating sub-acute ruminal acidosis (SARA). Sub-acute ruminal acidosis (SARA) occurs when highly fermentable carbohydrates are introduced into the diet, decreasing pH, and disturbing the microbial ecology of the rumen. Historically, antibiotics (e.g., monensin) have been used in the prevention and treatment of SARA. Biochanin A (BCA), an isoflavone produced by red clover (Trifolium pratense), mitigates microbial, end-product, and pH changes associated with starch fermentation in the laboratory. A pen study was conducted with ARS researchers and University of Kentucky collaborators to determine the effect of BCA in comparison to monensin or an untreated control on rumen bacteria and pH during a SARA challenge. BCA treatment counteracted the pH, microbiological, and fermentative changes associated with SARA challenge. Similar results were observed with monensin treatment except BCA was more effective at promoting fiber utilization. These results provide evidence that BCA may be an effective alternative for antibiotics in counteracting SARA when cattle are consuming high starch diets. The impact of this research is that beef producers could improve both cattle health and performance on high-starch diets through strategic supplementation with BCA or BCA containing legumes, like red clover.

Review Publications
Klotz, J.L. 2019. Ergocornine. Review Article. https://doi.org/10.1016/B978-0-12-801238-3.98305-3.
Greene, M.A., Klotz, J.L., Goodman, J.P., May, J.B., Harlow, B.E., Baldwin, W., Strickland, J.R., Britt, J.L., Schrick, F.N., Duckett, S.K. 2020. Evaluation of oral citrulline administration as a mitigation strategy for fescue toxicosis in sheep. Translational Animal Science. 4(4):txaa197. https://doi.org/10.1093/tas/txaa197.
Valente, E.E., Klotz, J.L., Ahn, G., Harmon, D.L. 2021. Pattern of postruminal administration of L-tryptophan affects blood serotonin in cattle. Domestic Animal Endocrinology. 74. Article 106574. https://doi.org/10.1016/j.domaniend.2020.106574.
Valente, E.E., Klotz, J.L., Harmon, D.L. 2020. 5-Hydroxytryptophan strongly stimulates serotoninsynthesis in Holstein steers. Domestic Animal Endocrinology. 74. Article 106560. https://doi.org/10.1016/j.domaniend.2020.106560.
Valente, E.E., Demasceno, M.L., Klotz, J.L., Harmon, D.L. 2021. Residual effects of abomasal 5-hydroxytryptophan administration on serotonin metabolism in cattle. Domestic Animal Endocrinology. 76. Article 106627. https://doi.org/10.1016/j.domaniend.2021.106627.
Greene, M.A., Britt, J.L., Bertrand, J., Klotz, J.L., Bridges, Jr., W.C., Andrae, J.G., Duckett, S.K. 2020. Feeding tall fescue seed during mid and late gestation influences subsequent postnatal growth, puberty, and carcass quality of offspring. Animals. 10(10). Article 1859. https://doi.org/10.3390/ani10101859.
Wilbanks, S.A., Justice, S., West, T., Klotz, J.L., Andrae, J.G., Duckett, S.K. 2021. Effects of tall fescue endophyte type and dopamine receptor D2 genotype on cow-calf performance during late gestation and early lactation. Toxins. 13(3). Article 195. https://doi.org/10.3390/toxins13030195.
Britt, J.L., Greene, M.A., Wilbanks, S.A., Bertrand, J., Klotz, J.L., Bridges, Jr., W.C., Aiken, G.E., Andrae, J.G., Duckett, S.K. 2020. Ergot alkaloid exposure during gestation: I. Impacts on ewe milk production, lamb birth weight and pre-weaning growth rate. Animals. 10(12). Article 2291. https://doi.org/10.3390/ani10122291.
Valente, E.E., Klotz, J.L., Ahn, G., McLeod, K.R., Herzing, H.M., King, M., Harmon, D.L. 2020. Ergot alkaloids reduce circulating serotonin in the bovine. Journal of Animal Science. 98(12):skaa362. https://doi.org/10.1093/jas/skaa362.
Seay, T.B., Melchior, E.A., Clemmons, B.A., Cordero, J.F., Bates, G.E., Flythe, M.D., Klotz, J.L., Ji, H., Goodman, J.P., McLean, K.J., Myer, P.R. 2020. Rumen and serum metabolomes in response to endophyte-infected tall fescue seed and isoflavone supplementation in beef steers. Toxins. 12(12). Article 744. https://doi.org/10.3390/toxins12120744.
Egert-McLean, A.M., Sama, M.P., Klotz, J.L., McLeod, K.R., Kristensen, N.B., Harmon, D.L. 2020. A moderate transition of beef heifers to a 90% concentrate diet as a model for subacute ruminal acidosis induced alterations in feeding behavior, rumen environment, reticulorumen motility, and blood acid-base status. Canadian Journal of Animal Science. 101(1):85-95. https://doi.org/10.1139/cjas-2019-0218.
McLean, K.J., Baldwin, R.L., Li, C., Klotz, J.L., Edwards, J.L., McLeod, K.R. 2020. Synthetic alkaloid treatment influences the intestinal epithelium and mesenteric adipose transcriptome in Holstein steers. Frontiers in Veterinary Science. 7. Article 615. https://doi.org/10.3389/fvets.2020.00615.