Use of 5-hydroxytryptophan to alleviate symptoms of fescue toxicosis in beef cattle

Abstract

The present disclosure concerns administration of 5-hydroxytryptophan (5-HTP) to animals, such as livestock, as an approach to inhibit and / or treat ergot alkaloid toxicosis in livestock. Acute exposure to ergots can be detrimental to livestock. The present disclosure shows that the administration of 5-HTP can counter the detrimental effects, normalizing both serotonin and feed intake for animals exposed to ergot alkaloids.

Description

USE OF 5-HYDROXYTRYPTOPHAN TO ALLEVIATE SYMPTOMS OF FESCUE TOXICOSIS IN BEEF CATTLE CROSS-RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application 63 / 618,059, filed January 5, 2024, the contents of which are hereby incorporated by reference in their entirety. GOVERNMENT SUPPORT

[0002] This invention was made with Government support under grant 201807121511 awarded by the United States Department of Agriculture Agricultural Research Service. The Government has certain rights in the invention. BACKGROUND

[0003] Ergot alkaloid intake can trigger many physiological dysfunctions that ultimately decrease cattle performance. Treatments to decrease the impact of ergot toxicosis on livestock productivity are needed in many countries. The molecular similarities between ergot alkaloids and biogenic amine neurotransmitters allows ergot alkaloids to interfere in the many functions associated with these neurotransmitters including serotonin. Ergot alkaloid receptor binding causes a long-term stimulation with serotonergic receptors become less responsive to serotonin after a period of exposure. Further, there is evidence that ergot alkaloids reduce circulating serotonin in cattle. It has also been reported that melatonin, a metabolite from the serotonin pathway, decreases in steers grazing endophyte-infected tall fescue. SUMMARY

[0004] The present disclosure concerns methods of administering hydroxylated tryptophan to counter ergot alkaloid toxicosis in livestock.

[0005] A 1staspect of the present disclosure, either alone or in combination with any other aspect herein concerns a method for inhibiting and / or treating ergot alkaloid toxicosis in an animal comprising administration to the animal of a composition comprising a hydroxylated typtophan.

[0006] A 2ndaspect of the present disclosure, either alone or in combination with any other aspect herein concerns the method of the 1staspect, wherein the tryptophan is an L- tryptophan.

[0007] A 3rdaspect of the present disclosure, either alone or in combination with any other aspect herein concerns the method of the 1stor 2ndaspect, wherein the trypthopan is hydroxylated at the 5 position of an indole therein.

[0008] A 4thaspect of the present disclosure, either alone or in combination with any other aspect herein concerns the method of the 1stor 2ndaspect, wherein the hydroxylated tryptophan is 5-hydroxytryptophan (5-HTP).

[0009] A 5thaspect of the present disclosure, either alone or in combination with any other aspect herein concerns the method of the 1staspect, wherein the animal is a livestock mammal.

[0010] A 6thaspect of the present disclosure, either alone or in combination with any other aspect herein concerns the method of the 5thaspect, wherein the animal is selected from the group consisting of a cow, an oxen, a bison, a buffalo, an ovine or caprine.

[0011] A 7thaspect of the present disclosure, either alone or in combination with any other aspect herein concerns the method of the 1staspect, wherein the animal is a bovine.

[0012] An 8thaspect of the present disclosure, either alone or in combination with any other aspect herein concerns the method of the 1staspect, wherein the composition is a food additive.

[0013] A 9thaspect of the present disclosure, either alone or in combination with any other aspect herein concerns the method of the 1staspect, wherein the composition is administered intravenously.

[0014] A 10thaspect of the present disclosure, either alone or in combination with any other aspect herein concerns the method of the 1staspect, wherein the composition is administered at about 0.1 to about 1.0 mg / kg of the animal’s weight.

[0015] An 11thaspect of the present disclosure, either alone or in combination with any other aspect herein concerns the method of the 10thaspect, wherein the composition is administered at 0.5 mg / kg of the animal’s weight.

[0016] A 12thaspect of the present disclosure, either alone or in combination with any other aspect herein concerns a method for alleviating toxicosis in an animal comprising administration to the animal of a composition comprising a hydroxylated typtophan.

[0017] A 13thaspect of the present disclosure, either alone or in combination with any other aspect herein concerns the method of the 12thaspect, wherein the tryptophan is an L- tryptophan.

[0018] A 14thaspect of the present disclosure, either alone or in combination with any other aspect herein concerns the method of the 12thor 13thaspect, wherein the trypthopan is hydroxylated at the 5 position of an indole therein.

[0019] A 15thaspect of the present disclosure, either alone or in combination with any other aspect herein concerns the method of the 12thor 13thaspect, wherein the hydroxylated tryptophan is 5-hydroxytryptophan (5-HTP).

[0020] A 16thaspect of the present disclosure, either alone or in combination with any other aspect herein concerns the method of the 12thaspect, wherein the animal is a livestock mammal.

[0021] A 17thaspect of the present disclosure, either alone or in combination with any other aspect herein concerns the method of the 16thaspect, wherein the animal is selected from the group consisting of a cow, an oxen, a bison, a buffalo, an ovine or caprine.

[0022] An 18thaspect of the present disclosure, either alone or in combination with any other aspect herein concerns the method of the 12thaspect, wherein the animal is a bovine.

[0023] A 19thaspect of the present disclosure, either alone or in combination with any other aspect herein concerns the method of the 12thaspect, wherein the composition is a food additive.

[0024] A 20thaspect of the present disclosure, either alone or in combination with any other aspect herein concerns the method of the 12thaspect, wherein the composition is administered intravenously.

[0025] A 21staspect of the present disclosure, either alone or in combination with any other aspect herein concerns the method of the 12thaspect, wherein the composition is administered at about 0.1 to about 1.0 mg / kg of the animal’s weight.

[0026] A 22ndaspect of the present disclosure, either alone or in combination with any other aspect herein concerns the method of the 21staspect, wherein the composition is administered at 0.5 mg / kg of the animal’s weight. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 shows dry matter intake of steers receiving ergot alkaloids (E+) and 5- hydroxytryptophan (5-HTP). Means (A) and over time (B), vertical dot line indicates when treatments started. Data are means ± SEM for N = 8 steers / group. P-value for E+ = 0.269, 5HTP+ = 0.0729, E+*5HTP = 0.003, Day <0.001.

[0028] Figure 2 shows serum prolactin of steers receiving ergot alkaloids (E+) and 5- hydroxytryptophan (5-HTP). Means (A) and over time (B), vertical dot line indicates when treatments started. Data are means ± SEM for N = 8 steers / group. P-value for E+ < 0.001, 5HTP = 0.303, E+*5HTP = 0.789.

[0029] Figure 3 shows changes in serum 5-hydroxytryptophan (5-HTP, A), serum serotonin (5-HT, B), plasma 5-HT (C) and 5-hydroxyindoleacetic acid (5-HIAA, D) in steers receiving ergot alkaloids (E+) and 5-hydroxytryptophan (5-HTP). Data are means ± SEM for N = 8 steers / group. P-value for (A) E+ = 0.957, 5HTP = <0.001, E+*5HTP = 0.550, Time = <0.001; (B) E+ = <0.001, 5HTP = 0.0729, E+*5HTP = 0.872, Time = 0.017; (C) E+ <0.001, 5HTP = 0.068, E+*5HTP = 0.347, Time = 0.017; (D) E+ = 0.394, 5HTP < 0.001, E+*5HTP = 0.242, Time < 0.001.

[0030] Figure 4 shows changes in serum tryptophan (A), serum kynurenine (B) and kynurenine x100 / tryptophan ration (KYN x 100 / TRP, C) in steers receiving ergot alkaloids (E+) and 5-hydroxytryptophan (5-HTP). Data are means ± SEM for N = 8 steers / group. A) E+ = 0.969, 5HTP = 0.002, E+*5HTP = 0.700, Time = 0.014; (B) E+ = 0.009, 5HTP < 0.001, E+*5HTP = 0.155, Time = 0.003; (C) E+ = 0.007, 5HTP = 0.002, E+*5HTP = 0.051, Time = 0.012.

[0031] Figure 5 shows changes in plasma glucose (A) and plasma non-esterified fatty acids (NEFA, B) in steers receiving ergot alkaloids (E+) and 5-hydroxytryptophan (5-HTP). Dataare means ± SEM for N = 8 steers / group. A) E+ = 0.581, 5HTP = 0.790, E+*5HTP = 0.202, Time = 0.004; (B) E+ = 0.609, 5HTP = 0.254, E+*5HTP = 0.202, Time < 0.001. DESCRIPTION

[0032] The present disclosure relates to the administration of 5-hydroxytryptophan (5- HTP) as an agent to mitigate ergot alkaloid-induced serotonin suppression. In some aspects, 5- HTP is administered to a bovine to treat and / or prevent serotonin suppression. In some aspects, 5-HTP is provided as a form of agonist therapy. Agonist therapy may refer to the administration of medications that share neurobiological mechanisms of an undesirable drug and can cause a neurochemical normalization. Agonist therapy has been shown as an effective treatment for neuromodulator drug dependence. A model agonist therapy for ergotism would be expected to normalize serotonin dysfunction. In an ex vivo study, serotonin showed potentialcompetition with ergot alkaloids for serotonergic receptors in bovine blood vessels. It i s possiblethat an increase of serotonin in the synaptic cleft could elevate competition with ergot alkaloids and reduce the overstimulation through a reduction in ergot alkaloid receptor association. However, the use of serotonin as therapeutic agonist for ergotism in cattle has not been reported.

[0033] Peripheral serotonin cannot cross the blood-brain barrier (BBB), yet administration of 5-HTP can be expected to enter the brain and presumably affect brainserotonin-mediated functions. As shown in the examples here in , t est s wereperf ormed t o assess whether 5-HTP administration can reduce corresponding adverseeffects associated with ergot alkaloids by increasing blood serotonin.

[0034] In some aspects, the present disclosure concerns the administration of 5-HTP to a bovine, such as a cow, an oxen, a bison, a buffalo, an ovine or caprine, such as a goat or a sheep, or similar mammal that is vulnerable to ergot alkaloids. In some aspects, the present disclosure concerns administration of L- and / or D- tryptophan that is modified with a hydroxyl group at the 5 position of the side chain’s indole. In some aspects, the present disclosure concerns the administration to a bovine with 5-hydroxy-L-tryptophan. In some aspects, the administration is by any route, such as oral, intravenous, intraperitoneal, dermal, sublingual,intramuscular, or combinations thereof. In some aspects, 5-HTP can be administered as a dietary supplement.

[0035] In some aspects, the present disclosure concerns administration of 5-HTP to a bovine orally. In some aspects, the present disclosure includes adding 5-HTP to a bovine’s diet, such as, as a supplement.

[0036] In some aspects, 5-HTP is administered at a dose of about 0.1 to about 1.0 mg / kg of the animal’s weight or mass. In some aspects, 5-HTP is administered at about 0.1 to 0.9, 0.1 to 0.8, 0.1 to 0.70.1 to 0.6, 0.1 to 0.5, 0.1 to 0.4, 0.1 to 0.3, 0.1 to 0.2, 0.2 to 1.0, 0.2 to 0.9, 0.2 to 0.8, 0.2 to 0.7, 0.2 to 0.6, 0.2 to 0.5, 0.2 to 0.4, 0.2 to 0.3, 0.3 to 1.0, 0.3 to 0.9, 0.3 to 0.8, 0.3 to 0.7, 0.3 to 0.6, 0.3 to 0.5, 0.3 to 0.4, 0.4 to 1.0, 0.4 to 0.9, 0.4 to 0.8, 0.4 to 0.7, 0.4 to 0.6, 0.4 to 0.5, 0.5 to 1.0, 0.5 to 0.9, 0.5 to 0.8, 0.5 to 0.7, 0.5 to 0.6, 0.6 to 1.0, 0.6 to 0.9, 0.6 to 0.8, 0.6 to 0.7, 0.7 to 1.0, 0.7 to 0.9, 0.7 to 0.8, 0.8 to 1.0, 0.8 to 0.9, and 0.9 to 1.0 mg / kg of the mass of the animal. For example, as set forth in the working examples herein, 5-HTP administered at about 0.5 mg / kg BW (bovine weight) attenuated some detrimental effects from ergot alkaloids. In particular, the ergot alkaloids show significantly higher receptor binding affinity over serotonin and its inhibitory effects can persist much longer than it is detectable. As set forth herein, administration of 5-HTP can be effective in normalizing serotonin, both in the plasma and serum of bovines.

[0037] In some aspects, 5-HTP can be administered as a single dose or repeated doses, such as 2, 3, 4, 5, 6, or 7 times a week, or further including multiple doses per day, such as two, three, four or more doses per day.

[0038] The present disclosure relates to a novel use of agonist therapy with a serotonin precursor to mitigate ergot alkaloid-induced toxicosis in cattle. The overstimulation of biogenic amine receptors by ergot alkaloids results in long-term effects that cause physiological changes with productive losses in livestock. The ergot alkaloids have significant negative economic impacts on livestock worldwide and the complex pharmacokinetics make it a challenge to develop an effective treatment. The 5-HTP administration in the present disclosure showed potential benefits to attenuating some of the detrimental effects of ergot alkaloids, specifically reduced DMI (dry matter intake) in cattle. This first study was only abrief look at the relationship, but if reductions in DMI could be alleviated, even partially, it could have a large impact on alleviating fescue toxicosis.

[0039] Ergot derivatives have slower receptor-association kinetics than serotonin. However, ergot alkaloids also have a slower receptor-dissociation rate and water washout resistance. This results in long-term receptor stimulation in many species including cattle that perpetuates the malady. Additionally, it seems that cellular internalization and desensitization could contribute to the prolonged effects of ergot alkaloids. Strategies to reduce ergot alkaloid receptor binding can potentially result in the mitigation of ergot toxicosis.

[0040] In contrast to serotonin, the ergot derivatives potency is time dependent. In an ex vivo study, the 24-h exposure of saphenous veins to ergovaline it was observed that ergovaline had a potency 5000-times higher than serotonin. Ergot alkaloids are extensively metabolized by the liver and cleared from the blood by first-pass hepatic metabolism observed a half-life of 23.6 min for ergovaline in sheep when solution with ergolvaline was infused into the jugular vein at the dose of 17 µg / kg. However, the ergot alkaloid effects can persist for much longer than it can be detected in plasma. Thus, it is desirable to utilize compounds that limit receptor binding by ergot alkaloids to accelerate clearance and to reduce the neuron excitation by ergot alkaloids and simultaneously treat the adverse effects.

[0041] It understood that ergot alkaloid consumption is associated with decreases in feed intake. However, the inhibitory effect of ergot alkaloids on DMI depends on dose, time of exposure and the presence of heat stress. Ergot alkaloids have molecular similarities with the biogenic amine neurotransmitters (e.g., norepinephrine, epinephrine, dopamine and serotonin) allowing them to bind to these receptors and interfere in the many associated functions. Based on the improvement seen in DMI for E+ / 5-HTP+ as set forth herein, much of the observed reduction in DMI may be attributed to acting through serotonergic receptors.

[0042] Serotonin in the brain influences appetite and body weight via the 2C and 1B serotonin receptor subtypes. Therefore, the overstimulation of serotonergic receptors by ergot alkaloids can cause reductions in feed intake. The administration of 5-HTP in association with ergovaline is effective to normalize the DMI. The possible increase of serotonin in neuron clefts caused by the 5-HTP infusion may be responsible for increased receptor competition and reductions in E+ binding to receptors. Although high doses of 5-HTP havecaused suppression of food intake in rats and cattle, moderate doses of 5-HTP increase circulating serotonin without suppressing DMI in cattle.

[0043] The reduction of serum prolactin with the administration of E+ is evidence that the induction of ergot alkaloids toxicosis was achieved. Serum prolactin is commonly utilized as an indicator of ergot alkaloid toxicosis. Ergot alkaloids can reduce prolactin by acting as a dopamine agonist which has inhibitory effects on prolactin synthesis. Although there are interactions between serotonin and dopamine transporters, it seems unlikely that increased serotonin will prevent ergot alklaoids from binding to dopaminergic receptors.

[0044] The decrease of serotonin after consumption of E+ can be caused by a feedback in response to the overstimulation of serotonergic neurons. This metabolic behavior in cattle has been previously observed. The chronic neural stimulation causes long-term changes in neurochemistry. Increases of serotonin in a neuronal cleft activates autoreceptors which may provoke actions to decrease the firing rate. Although serotonin is controlled and metabolized by several metabolic pathways, the ergot alkaloids do not have this control mechanism and may induce a decrease of serotonin in a physiological attempt to reduce overstimulation of the serotonergic neurons.

[0045] Serotonin can be removed from the synaptic cleft by binding to a selective serotonin reuptake transporter that transports it into the presynaptic cytosol to terminate signaling. Once there, the serotonin is repackaged into vesicles for storage or metabolized by monoamine oxidase producing 5-hydroxyindoleacetic acid, which is excreted into urine (Billett, 2004). The catabolism of serotonin is a mechanism to diminish serotonergic neuron signalling and it is controlled by the activity of monoamine oxidase (Scotton et al., 2019). However, the 5-hydroxyindoleacetic acid, a metabolite from serotonin catabolism, was not affected by E+ in this study. The delayed peak for serum 5-hydroxyindoleacetic acid following the E+ / 5HTP+ treatment, in comparison to E- / 5HTP+, may be caused by an attempt to reestablish homeostasis through increasing the serotonin storage following the depletion by E+. Once the serotonin was repleted, the degradation pathway appeared to increase and a peak of 5-hydroxyindoleacetic acid occurred at the second hour after infusion.

[0046] Therefore, the control of serotonin degradation seems to not be the main reason for the observed depletion of serotonin associated with ergot alkaloids. It is more likelythat the decreased serotonin caused by E+ was related to a reduction in synthesis than an increase in degradation. Autoreceptors on terminals decrease synthesis and release when extracellular serotonin rises. Serotonin is synthesized from trypthophan through hydroxylation and decarboxylation catalyzed by the enzymes tryptophan hydroxylase and the aromatic acid decarboxylase, respectively. The availability of those enzymes is limiting the synthesis of serotonin. The autoreceptors can reduce the activity of tryptophan hydroxylase and consequently, the serotonin synthesis.

[0047] Infusion of 5-HTP was efficient in normalizing both serum and plasma serotonin. Plasma serotonin is related to free serotonin in the blood while the serum serotonin is related to total circulating concentration which is the sum of free and platelet serotonin. The conversion of 5-HTP is catalyzed by L-amino acid decarboxylase enzyme, which is widely distributed and it is not rate-limiting under normal conditions. The normalization of circulating serotonin in the steers receiving E+ after 5-HTP reinforces the hypothesis that ergot alkaloids reduce serotonin synthesis.

[0048] Prolonged stimulation of serotonergic neurons desensitizes the receptors. Depletion of serotonin in rats induced by p-chlorophenylalanine, an inhibitor of tryptophan hydroxylase, resulted in the supersensitivity to lysergic acid. Therefore, it is possible that the serotonin normalization can contribute to a reduced potency of ergot alkaloids.

[0049] The increase of 5-HTP seems to alter tryptophan metabolism. The 5- HTP administration showed a clear reduction in serum tryptophan and kynurenine in the first hours after administration. The decrease of kynurenine after 5-HTP infusion had been observed before. However, it is not clear how 5-HTP affects kynurenine concentration. The serotonin and kynurenine pathways are competitive and controlled by analogous enzymes. Therefore, a decrease in the kynurenine pathway can be favorable to increases in serotonin synthesis.

[0050] Although serotonin did not change glucose and NEFA in this study, it is recognized that serotonin has a role in the regulation of energy balance, specifically in relation to NEFA and glucose metabolism. Results for 5-HTP dosing on energy metabolism in cattle has been diverse with experiments demonstrating an increase of glucose, increase of insulin (Field et al., 2021), no effect on glucose and decrease in insulin.

[0051] The potential of 5-HTP to mitigate ergot alkaloid toxicosis can be related to receptor binding dynamics. Evidence suggests that increases in synaptic serotonin can reduce the stimulant properties of some drugs and can be used during withdrawal treatments. The increase of serotonin and receptor competition may reduce the firing of serotonergic neurons by ergot alkaloids. Agonist medications have to share pharmacodynamic mechanisms of action with the undesired molecule but usually have distinct pharmacokinetic characteristics. Serotonin has a dissociation rate considerably higher than ergot derivatives indicating its potential as an agonist for ergotism.

[0052] Ergot alkaloids accordingly reduce DMI and circulating serotoninconcentrations. Conversely, post-ruminal 5-HTP administration increases the blood concentration of serotonin and offsets the lower DMI caused by E+ with no evidence of effects on energy metabolism. Therefore, administration of 5-HTP has potential to be used to normalize feed intake and circulating serotonin in cattle exposed to ergot alkaloids. Future research will focus on the evaluation of thermoregulation in association with ergotism and 5-HTP supplementation along with an assessment in a larger population over extended periods. EXAMPLES

[0053] Material and Methods

[0054] Animals, design and treatment

[0055] Eight Holstein steers (538 ± 18 kg) fitted with ruminal cannulas (Bar Diamond, Inc., Parma, ID, USA) were used in a replicated 4 x 4 Latin Square design experiment with a 2 x 2 factorial treatment structure. The treatments were the combination of 0 (E-) or 15 µg ergovaline / kg BW (E+) and 0 (5HTP-) or 0.5 mg of 5-hydroxy-L-tryptophan / kg BW (5HTP+). The ergovaline (E+) and 5-hydroxy-L-tryptophan (5-HTP) were administrated from day 1 to day 6 of each period. The steers received a complete mixed diet (Table 1) once a day at 0800 h allowing for 5% orts.

[0056] Toxic endophyte-infected tall fescue seed was used to supply the daily dose of 15 μg / kg BW ergovaline. The treatment with 0 μg / kg BW ergovaline received endophyte-free seed at the same amount as E+ to equalize seed intake. Seeds were ground and placed into rumen immediately before feeding. The amount and method of infusion the 5-HTP was chosenbased upon a previous study (Valente et al., 2021a). Pure 5-HTP (97%; Bulk supplements, Henderson, Nevada, USA) was abomasally infused in a single dose following seed dose. The 5-HTP was dissolved in 50 mL of tap water before the infusion. After the 5-HTP infusion, 50 mL water was infused to purge the infusion line of any remaining 5-HTP. The dosage was calculated based on individual body weight, measured, and adjusted each period. The solution was infused into the abomasum at a rate of about 100 mL / min using a syringe.

[0057] Intra-abomasal infusions were accomplished by placing an infusion line made of flexible 6.3 mm o.d. tubing (Tygon®) and a plastisol washer (60 mm o.d.) through the reticulo-omasal orifice into the abomasum. The omasal pillar was used as a landmark and the washer was manipulated until it was distal to the pillar. The infusion line was inserted at the beginning of the each period and remained until all samples were collected.

[0058] All steers were housed indoors in individual pens (3 x 3 m) each with its own feed bunk and water supply. A thermoneutral status was maintained at 22 °C with a light:dark cycle of 16 h light and 8 h dark. The steers had an initial 10 d for acclimation to facilities and the basal diet. Each period lasted 6 d with a 15-d washout between treatment periods. Body weight was measured before feeding at the beginning of each period.

[0059] Blood sampling

[0060] Temporary i.v. catheters (14 Ga x 13 cm, Mila International, Inc., Florence, KY, USA) were inserted into the jugular vein one day before the blood sampling. Blood was collected from the jugular vein using catheters at 0, 1, 2, 4, 8 and 24 h after seed dosing and 5- HTP infusion on day 6. Before blood collection, 5 mL of blood and lock solution were aspirated from the catheter and discarded. Then a 20-mL sample of blood from the jugular catheter was collected, 16 mL was transferred to two 10 mL tubes, one containing heparin and the other with clot activator, 4 mL was transferred to a 6 mL tube with sodium fluoride.

[0061] After blood collection, a lock solution of Na4EDTA (30 mg / mL) was infused (3 mL)into thecatheter to flush blood and maintain patency. Blood samples for serum were kept at room temperature for 45-60 min for clotting before centrifugation. Blood samples for plasma were immediately centrifuged after collection at 5000 x g for 10 min at and samples forserum were centrifuged after clotting at 5000 x g for 10 min at 15 ºC. Plasma and serum were transferred to 1.5 mL vials and kept at -80until analysis.

[0062] Biochemical analysis

[0063] Using the procedures of Bernard et al. ( 1993) serum prolactin analysis was conducted by the laboratory of J. L. Edwards (University of Tennessee, Knoxville) utilizing radioimmunoassay with average intra-assay and inter-assay coefficients of variation of 4.14 and 8.36%, respectively. Because of the inherent instability, serotonin and its metabolites were analyzed between 24 and 72 h after sampling. The 5-HTP, 5-hydroxyindoleacetic acid, tryptophan, and kynurenine concentrations were analyzed in the serum. Serotonin concentrations were evaluated in the serum (total) and the plasma (free). Glucose and free fatty acids (FFA) were analyzed only in plasma (containing sodium fluoride).

[0064] The 5-HTP, 5-hydroxyindoleacetic acid, tryptophan, serotonin, and kynurenine were simultaneously measured with a high-performance liquid chromatography (HPLC) method modified from Sa et al. (2012). Briefly, an HPLC (model 2695; Waters, Milford, Massachusetts, EUA) with a fluorescence detector (model 2475; Waters, Milford, Massachusetts, EUA) and an ultraviolet detector (Model 2487; Waters, Milford, Massachusetts, EUA) were used.

[0065] The fluorescence detector was used with excitation wavelength set at 278 nm and the emission wavelength at 338 nm for 5-HTP, 5-hydroxyindoleacetic acid, tryptophan, and serotonin while the ultraviolet detector was set at 365 nm for kynurenine analysis. The chromatographic separation was carried out using a ACE C18-PFP column (4.6×150 mm, 3 μm;, Advanced Chromatography Technologies,Aberdeen, Scotland) at 40 ºC.The mobile phase consisted of 0.05 M KH2PO4and methanol(85:15, v / v; pH = 4.3), at a flowrate of 1.0 mL / min. Serum and plasma samples were deproteinized by mixing with an equal volume of 5% (v / v) perchloric acid, followed by vortexing and centrifuging at 20,000 × g for 10 min at 4 °C. The supernatant was diluted four-fold with ultrapure water and centrifuged at 20,000 × g for 10 min at 4 °C. Ultimately, 10 μL of the supernatant was injected into the HPLC for analysis.

[0066] Glucose and non-esterified fatty acids (NEFA) concentrations were analyzed using an automatic analyzer (Konelab 20XTI; Thermo Electron Corporation) using commercial kits. Glucose was analyzed using the Glucose Hexokinase Infinity Reagent (Infinity TM, Thermo Fisher Scientific, Waltham, MA) and NEFA were analyzed using Wako HR series NEFA-HR (Fujifilm, Lexington, MA).

[0067] Feed Intake

[0068] The offered feed was adjusted daily according to the orts from the previous day, keeping orts at approximately 5%. Feed intake was recorded every day. The feed and orts samples were analyzed for dry matter (DM, index 920.39) according to AOAC (1990). The DM intake before infusion was calculated as the average of four days before infusion and used as baseline. The first four days after E+ and 5-HTP administration were used as response to the treatments because on day 5 jugular catheters were inserted and caused a disturbance of normal feeding.

[0069] Statistical Analysis

[0070] All data were analyzed as completely randomized designs using the MIXED model of SAS (OnDemand version, 2021; SAS Inst. Inc. Cary, NC):

[0071] Where, Yijk is the dependent variable, µ is the overall mean, Si is the effect of steer, Pj is the effect of period, LSk is the effect of Latin Square, E+l is the effect of the ergovaline treatment, 5HTPm is the effect of the 5-hydroxytryptophan treatment, E+*5HTPlm is the effect of the interaction between E+ and 5-HTPlm and eijklm is the random error.

[0072] The area under the curve (AUC) was calculated using the linear trapezoidal method. Shapiro and Wilk's (1965) test for normality was performed using procedure Univariate of SAS. The 5-HTP and serotonin data were transformed according Box and Cox (1964) because of the lack of normality as following:^^^^ =^ , ^^ ^ ≠ 0logy, ^^ ^ = 0

[0073] The lambda for each variable was calculated using SAS Transreg with model boxcox ranging from -2 to 2. After statistical analysis, the data were back-transformed to concentration for presentation. The standard error (SE) of the back-transformed data was calculated from the confidence limits of the transformed data as follows: upper CL − lower CLSE =3.92 Where upper CL is back-transformed upper confidence limit and lower CL is back- transformed lower confidence limit.

[0074] The feed intake and blood metabolites were analyzed as repeated measurements over time. Blood concentration at time zero was used as covariate. Various covariance structures of errors were fitted; the variance components structure (VC) was selected based on the lowest Bayesian information criterion (BIC). Pairwise comparisons were conducted using the LSD test. Statistical significance was considered at P ≤ 0.05 and tendency was considered at 0.05 < P ≤ 0.10.

[0075] Steers remained heathy throughout the experiment and gained on average 1.4 ± 0.47 kg / d. Chemical composition of the feeds was unaffected by seed additions (Table 1). There was an interaction (P < 0.05) between E+ and 5-HTP administration on dry matter intake (DMI). The E+ / 5HTP- decreased (P < 0.05) DMI in comparison to E- / 5HTP-. However, E+ / 5HTP+ normalized the DMI. The E- / 5HTP+ had similar (P > 0.05) DMI to E- / 5HTP- (Figure 1). Table 1- Diet composition fed to steers for the study of administration of ergot alkaloids (E+) and intra-abomasal 5-hydroxytryptophan on serotonin metabolism Item Acclimation diet E- diet E+ diet Ingredient (g / kg, DM) Corn silage 750 670 671 Corn grain cracked 120 103 103 Distillers grains with solubles 30 - - Corn ground grain 69.7 62 62 Limestone 19.2 15 15 Premix15 4 4 Urea 3.6 3 3 Choice white grease 2.5 2 2 Tall fescue KY32 - 141 -Tall fescue 3rd Millennium - - 140 Chemical composition (g / kg, DM) Crude protein 101 104 102 Total digestible nutrients 725 730 7301Commercial product (Kentucky Nutrition Service, Lawrenceburg, KY, USA): NaCl (920-960 g / kg), Fe (92.8 g / kg), Zn (55 g / kg), Mn (47.9 g / kg), Cu (1835 ppm), I (115 ppm), Se (18 ppm) and Co (65 ppm).

[0076] There was no interaction between E+ and 5-HTP for serum prolactin (P > 0.05). Steers receiving E+ had lower (P < 0.005) serum prolactin (Figure 2). The 5-HTP did not change (P > 0,05) serum prolactin in steers receiving E- or E+. There was no interaction between administration of E+ and 5-HTP (P > 0.05) for area under curve (AUC) of blood metabolites (Table 2). Although E+ did not affect the AUC of serum 5-HTP, 5-hydroxyindoleacetic acid, tryptophan and kynurenine, serum and plasma concentration of serotonin were all decreased (P < 0.05). The infusion of 5-HTP increased (P < 0.05) the AUC of serum 5-HTP, serum and plasma serotonin and serum 5-hydroxyindoleacetic acid. The 5-HTP had no effect (P > 0.05) on AUC of serum tryptophan, but decreased (P < 0.05) serum kynurenine. In comparison with E- / 5HTP-, the E+ / 5HTP- decreased (P < 0.05) AUC from both serum and plasma serotonin while the E+ / 5-HTP+ had similar (P > 0.05) values. Table 2 - Area under curve for serum and plasma metabolites related to serotonin metabolism in steers receiving ergot alkaloids (E+) and 5-hydroxytryptophan (5HTP) Item1E- E+ SEM P-value 5HTP- 5HTP+ 5-HTP- 5HTP+ E+ 5HTP E+*5HTP Serum 5-HTP 2.77 4.60* 2.88 4.72* 0.38 0.577 <0.001 0.953 5-HT 140.4 161.5 99.7* 142.1 24.8 0.019 0.014 0.374 5-HIAA 0.99 3.14* 0.93 2.77* 0.66 0.345 <0.001 0.494 Tryptophan 872.2 840.4 885.2 847.8 48.9 0.773 0.334 0.934 Kynurenine 137 125.3 136.9 122.9* 6.1 0.840 0.010 0.870 Plasma 5-HT 9.72 14.67* 4.56* 7.36 2.55 <0.001 <0.001 0.427 Glucose 103.4 102.3 106.5 103.8 2.88 0.337 0.431 0.729 NEFA 1.69 1.83 1.63 1.74 0.16 0.407 0.192 0.88615-HTP = 5-hydroxytryptophan; 5-HT = serotonin; 5-HIAA = 5-hydroxyindoleacetic acid; NEFA=non-esterified fatty acids. *Indicates a difference relative to E- / 5HTP- (P < 0.05).

[0077] Serum 5-HTP, serotonin and 5-hydroxyindoleacetic acid were (P < 0.05) affected by time while plasma serotonin did not change over time (Figure 3). The serum 5-HTP had a peak at 2 h after infusion of 5-HTP independently of the presence of E+ with the concentration returning near baseline after 8 h (Figure 3A). The increase of serotonin in response to 5-HTP was more intense in the plasma than serum (Figure 3B, C). In contrast to serum 5-HTP, serum and plasma serotonin had a steady increase without a clear peak. The serum 5- hydroxyindoleacetic acid peaked at 1h after infusion in the E- / 5HTP+, and about 2- h in the E+ / 5HTP+ (Figure 3D).

[0078] The AUC for serum tryptophan had no effect (P > 0.05) of E+ or 5-HTP (Table 2). Serum kynurenine had a lower (P < 0.05) AUC in the steers that received 5-HTP, with no effect (P > 0.05) of E+ (Table 2). Serum tryptophan and kynurenine concentrations decreased (P < 0.05) after 2 to 4 h after 5-HTP infusion (Figure 4A, B). Although tryptophan was no affected by E+, the kynurenine and kynurenine:tryptophan ratio was lower (P < 0.05; Figure 4B, C).

[0079] The AUC of plasma glucose and NEFA were unaffected (P > 0.05) by the administration of E+ or 5-HTP (Table 1). Plasma concentration of glucose and NEFA changed with time (P < 0.05), but were not affected (P > 0.05) by the administration of E+ or 5-HTP (Figure 5).

[0080] Various modifications of the present disclosure, in addition to those shown and described herein, will be apparent to those skilled in the art of the above description. Such modifications are also intended to fall within the scope of the appended claims.

[0081] It is appreciated that all reagents are obtainable by sources known in the art unless otherwise specified.

[0082] It is also to be understood that this disclosure is not limited to the specific aspects and methods described herein, as specific components and / or conditions may, of course, vary. Furthermore, the terminology used herein is used only for the purpose of describing particular aspects of the present disclosure and is not intended to be limiting in any way. It will be also understood that, although the terms “first,” “second,” “third” etc. may be used herein to describe various elements, components, regions, layers, and / or sections, these elements, components, regions, layers, and / or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer, or section from anotherelement, component, region, layer, or section. Thus, “a first element,” “component,” “region,” “layer,” or “section” discussed below could be termed a second (or other) element, component, region, layer, or section without departing from the teachings herein. Similarly, as used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms, including “at least one,” unless the content clearly indicates otherwise. “Or” means “and / or.” As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. It will be further understood that the terms “comprises” and / or “comprising,” or “includes” and / or “including” when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof. The term “or a combination thereof” means a combination including at least one of the foregoing elements.

[0083] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0084] Reference is made in detail to exemplary compositions, aspects and methods of the present disclosure, which constitute the best modes of practicing the disclosure presently known to the inventors. The drawings are not necessarily to scale. However, it is to be understood that the disclosed aspects are merely exemplary of the disclosure that may be embodied in various and alternative forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for any aspect of the disclosure and / or as a representative basis for teaching one skilled in the art to variously employ the present disclosure.

[0085] Patents, publications, and applications mentioned in the specification are indicative of the levels of those skilled in the art to which the disclosure pertains. These patents, publications, and applications are incorporated herein by reference to the same extent as if eachindividual patent, publication, or application was specifically and individually incorporated herein by reference.

[0086] The foregoing description is illustrative of particular embodiments of the disclosure, but is not meant to be a limitation upon the practice thereof. The following claims, including all equivalents thereof, are intended to define the scope of the disclosure.

Claims

CLAIMS 1. A method for inhibiting and / or treating ergot alkaloid toxicosis in an animal comprising administration to the animal of a composition comprising a hydroxylated typtophan.

2. The method of claim 1, wherein the tryptophan is an L-tryptophan.

3. The method of claim 1 or 2, wherein the trypthopan is hydroxylated at the 5 position of an indole therein.

4. The method of claim 1 or 2, wherein the hydroxylated tryptophan is 5-hydroxytryptophan (5-HTP).

5. The method of claim 1, wherein the animal is a livestock mammal.

6. The method of claim 5, wherein the animal is selected from the group consisting of a cow, an oxen, a bison, a buffalo, an ovine or caprine.

7. The method of claim 1, wherein the animal is a bovine.

8. The method of claim 1, wherein the composition is a food additive.

9. The method of claim 1, wherein the composition is administered intravenously.

10. The method of claim 1, wherein the composition is administered at about 0.1 to about 1.0 mg / kg of the animal’s weight.

11. The method of claim 10, wherein the composition is administered at 0.5 mg / kg of the animal’s weight.

12. A method for alleviating toxicosis in an animal comprising administration to the animal of a composition comprising a hydroxylated typtophan.

13. The method of claim 12, wherein the tryptophan is an L-tryptophan.

14. The method of claim 12 or 13, wherein the trypthopan is hydroxylated at the 5 position of an indole therein.

15. The method of claim 12 or 13, wherein the hydroxylated tryptophan is 5- hydroxytryptophan (5-HTP).

16. The method of claim 12, wherein the animal is a livestock mammal.

17. The method of claim 16, wherein the animal is selected from the group consisting of a cow, an oxen, a bison, a buffalo, an ovine or caprine.

18. The method of claim 12, wherein the animal is a bovine.

19. The method of claim 12, wherein the composition is a food additive.

20. The method of claim 12, wherein the composition is administered intravenously.

21. The method of claim 12, wherein the composition is administered at about 0.1 to about 1.0 mg / kg of the animal’s weight.

22. The method of claim 21, wherein the composition is administered at 0.5 mg / kg of the animal’s weight.

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