Performing Department
(N/A)
Non Technical Summary
Annual economic loss for the United States beef industry due to tall fescue toxicosis was estimated to be $2 billion, of which $500 million was due to decreased growth of calves consuming tall fescue containing ergot alkaloids. Ergot alkaloids decrease feed intake, increasevasoconstriction of core and peripheral blood vessels, and possibly contribute to immune system activation. The overall goal of this project is to evaluate the potential for 5-hydroxytryptophan (5-HTP) to ameliorate fescue toxicosisin beef cattle. The objectives of this project are to determine: 1) if increasing serum serotonin (5-HT) with 5-HTP restores feed intake, 2) if increasing serum 5-HT with 5-HTP causes vasodilation of ergot alkaloid-constricted blood vessels and reduces susceptibility to heat stress, 3) if alleviating ergot alkaloid-induced vasoconstriction and heat stress are associated with repairing compromised immune system function and gut integrity, and 4) identifying genes and pathways associated with the onset and amelioration of fescue toxicosis by ergot alkaloids and 5-HTP, respectively.It is anticipated that this project will lead to the first report demonstrating that: increased serum 5-HT from 5-HTP will lead to increased vasorelaxation, restored dry matter intake, and decreased immunoactivation andRNA-Seq analysis of blood vessels, hypothalamus, and intestinal tissue will identify genes, pathways, and functions associated with these phenotypic responses. This new knowledge will aid in efforts to develop strategies to ameliorate fescue toxicosis in grazingbeef cattle.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Goals / Objectives
The goal of this project is to evaluate the potential of 5-hydroxytryptophan to ameliorate fescue toxicosis in beef cattle and elucidate molecular mechanisms associated with the onset and amelioration of fescue toxicosis. The specific objectives of the project plan are:To determine the effects of ergot alkaloids and 5-hydroxytryptophan on feed intake regulation and feeding behaviorTo evaluate the potential of5-hydroxytryptophan to reduce ergot alkaloid-induced vasoconstriction and susceptibility to heat stressTo characterize the effects of ergot alkaloids and 5-hydroxytryptophan on gastrointestinal permeability and systemic inflammationTo identify genes, pathways, and functions affected by ergot alkaloids and5-hydroxytryptophan in the hypothalamus, lateral saphenous vein, and jejunal mucosa using RNA-Seq?
Project Methods
Crossbred beef steers (n=24; BW=250 kg) will be ruminally cannulated for use in a randomized complete block design experiment. Six blocks (replications of the experiment) will be conducted due to the limited time to assess viable blood vessels for myography, assess arterial hemodynamics, and sample blood. Steers will be housed indoors in individual pens (3 × 3 m) at the UK C. Oran Little Research Center. To mimic summer conditions, the room will have a 16:8 h light-dark cycle and room temperature will be increased to 32°C during the light and decreased to 21°C during the dark. Steers will be fed alfalfa cubes to supply 1.5 × the net energy required for maintenance plus a mineral supplement. Steers will be randomly assigned to one of four treatments: 1) non-toxic novel endophyte-infected tall fescue seed (NTE; control), 2) NTE + 5-HTP, 3) TE, or 4) TE + 5-HTP. Ground NTE (Lacefield MaxQ II) or TE (KY-31) seed will be dosed through the rumen cannula before feeding. The dose of TE will provide 15 mg ERV/kg BW/d which has been shown to induce effectively fescue toxicosis and reduce serum 5-HT concentration by 48% (1, 21). TE and NTE treatments will be balanced to consume the same amount of seed daily. The source of 5-HTP will be 5-HTP powder from griffonia seed extract (97% purity; Bulk Supplements). The dose of 5-HTP will be 3.3 mg 5-HTP/kg of BW/d, as this dose was shown to increase serum 5-HT by 60% without reducing DMI in our preliminary experiment. The dose of 5-HTP will be ruminally-dosed along with the fescue seed treatments. Treatments will be administered for 14-d, as this is sufficient to observe a decrease in serum prolactin in steers consuming TE and an increase in serum 5-HTP with 5-HTP. The following day (day 15), steers will be slaughtered at the UK Meats Laboratory for tissue collection.Dry matter intake will be measured daily. Feed and orts will be collected daily and analyzed for DM content. DMI distribution throughout the day will be monitored every minute by an electronic scale with datalogger attached to each animal's feed bunk (Campbell Scientific, Logan, Utah, United States). Feeding behavior (frequency of meals, meal size, and meal duration) will be calculated.In addition to DMI, core temperature, skin temperature, rectal temperature, and respiration rateare commonly measured to be used as indicators of heat stress. Each steer will have a radio telemetry device (CorTemp, HQ Inc.) placed in the abdomen (at the time of rumen fistulation) to continually measure and record core temperature at 1-min intervals. Skin temperature will be measured in the neck region with an infrared thermometer. Rectal temperature will be measured using a digital thermometer. Respiration rate will be measured over 5-min intervals. Measurements will be taken daily at 700 h and 1500 h.Caudal artery hemodynamics at the 4thcoccygeal vertebrae will be measured using color Doppler ultrasonography (Terason 3300 Portable Ultrasound Scanner) as described previously. Measurements will be taken 5 h after the morning feeding on days -1 (baseline), 1, 7, 14. Three cardiac cycle waveforms from three separate scans will be recorded and averaged for each blood vessel. Preset functions of the ultrasound instrument will be used to obtain systolic and diastolic velocity, pulsatility index, resistance index, and cross-sectional area of vessel. Mean velocity will be calculated as the difference between systolic and diastolic velocity, divided by the pulsatility index. Blood flow will be calculated as mean velocity × cross-sectional area × 60 s.On days 1, 7, and 14 of the treatment period, blood (30 mL) will be collected from the jugular vein with a syringe at 0 (before feeding and treatment administration) and 4-h after feeding. Blood will be stored in K2EDTA tubes or tubes with clot activator. During blood sampling, steers will continue to have access to feed and water. Blood will be centrifuged at 2000 × g for 20 min at 4°C to recover serum and plasma, aliquoted into 2-mL tubes, and stored at -80°C. Plasma biomarkers of energy metabolism and intake regulation (glucose, urea-N, NEFA, BHB, insulin), tryptophan metabolism (5-HTP, 5-HT, 5-hydroxyindoleacetic acid, tryptophan, kyurenine), and inflammation (citrulline, lactate, serum amyloid A, LPS binding protein, haptoglobin) will be measured.On day 13, total-tract gastrointestinal barrier function will be assessed using CrEDTA as a paracellular permeability marker. Before feeding, 500 mL of CrEDTA solutionwill be administered through the ruminal cannula. Steers will be fitted with temporary jugular catheters and blood will be collected at 0, 1, 2, 4, 8, 12, 18, and 24 h relative to CrEDTA administration and processed as described previously. Chromium concentration will be determined using atomic absorption spectroscopy (Aanalyst 200; Perkin Elmer Inc.).At slaughter, the hypothalamus, LSV, and jejunal mucosawill be rapidly collected in RNAlater to preserve tissues for RNA-Seq analysis. RNA-Seq will be conducted to identify target genes, pathways, and functions that are associated with the onset and alleviation of fescue toxicosis symptoms by EA and 5-HTP. RNA extraction, quantification, integrity, and sequencing will be conducted according to methods described in our laboratory previously.Strand-specific cDNA library preparation and RNA sequencing will be conductedto generate 150 bp paired-end reads at a depth of 20 M reads in each direction. Differential gene expression analyses, enrichment analyses, and statistical analyses will be conducted as previously described.The viscera will be removed from the body cavity, organs will be separated, and digesta will be removed to obtain tissue weights. For morphology measurements, intestinal cross-sections will be placed into tissue cassettes and submerged in 10% phosphate-buffered formalin. Formalin-fixed tissues will be dehydrated and embedded in paraffin. Blocks will be sectioned (5-μm thick), mounted with each slide containing 2 to 3 sections, and stained with hematoxylin and eosin. Villus height, villus width, crypt depth, and crypt width will be evaluated in at least 15 well-oriented crypt-villus units using an Axiovert microscope (Carl Zeiss Inc.) and imaging software (Scion Corporation). Morphology measurements will be used to calculate mucosal surface area index (89). For immunohistochemistry, intestinal cross sections will be prepared for sectioning and staining using the STAINperfect immunostaining kit A (ImmuSmol). Slides will be stained with rabbit anti-serotonin polyclonal primary antibody (IS1007; ImmuSmol) and goat anti-rabbit AlexaFluor 488 secondary antibody (A11034; ThermoFisher Scientific Inc.). 5-HT-positive area and the number of 5-HT positive cells in each tissue section will be determined using an Axioscan Z1 slide scanner.Cranial branches of LSV will be collected as a model of peripheral vasculatureto assess effects ofin vivoEA and 5-HTP onex vivocontraction and relaxation potential to 5-HT. Isolated LSV segments (2-mm) will be placed into chambers of a multi-myograph system (Danish Myo Technology 610M) with 5 mL of continuously oxygenated Krebs-Henseleit buffer. Vessels will be equilibrated for 90 min to 1 g tension. For the contraction experiment, LSV segments will be exposed to increasing concentrations of 5-HT (1 × 10-9to 1 × 10-4M) for 5 min. For the relaxation experiment, phenylephrine (1 × 10-4M) will be added for 15 min to pre-contract the blood vessel and then LSV segments will be exposed to the same 5-HT concentrations used in the contraction experiment for 5 min. Maximal vascular 5-HT-induced contraction and relaxation will be quantified and normalized for each blood vessel.