**Rumen and Serum Metabolomes in Response to Endophyte-Infected Tall Fescue Seed and Isoflavone Supplementation in Beef Steers**

**Taylor B. Ault-Seay 1 , Emily A. Melchior-Ti**ff**any 1,**† **, Brooke A. Clemmons 1,**‡ **, Juan F. Cordero 1 , Gary E. Bates 2 , Michael D. Flythe 3 , James L. Klotz 3 , Huihua Ji 4 , Jack P. Goodman 5 , Kyle J. McLean <sup>1</sup> and Phillip R. Myer 1, \***


Received: 4 November 2020; Accepted: 24 November 2020; Published: 26 November 2020

**Abstract:** Fescue toxicosis impacts beef cattle production via reductions in weight gain and muscle development. Isoflavone supplementation has displayed potential for mitigating these effects. The objective of the current study was to evaluate isoflavone supplementation with fescue seed consumption on rumen and serum metabolomes. Angus steers (*n* = 36) were allocated randomly in a 2 × 2 factorial arrangement of treatments including endophyte-infected (E+) or endophyte-free (E−) tall fescue seed, with (P+) or without (P−) isoflavones. Steers were provided a basal diet with fescue seed for 21 days, while isoflavones were orally administered daily. Following the trial, blood and rumen fluid were collected for metabolite analysis. Metabolites were extracted and then analyzed by UPLC-MS. The MAVEN program was implemented to identify metabolites for MetaboAnalyst 4.0 and SAS 9.4 statistical analysis. Seven differentially abundant metabolites were identified in serum by isoflavone treatment, and eleven metabolites in the rumen due to seed type (*p* < 0.05). Pathways affected by treatments were related to amino acid and nucleic acid metabolism in both rumen fluid and serum (*p* < 0.05). Therefore, metabolism was altered by fescue seed in the rumen; however, isoflavones altered metabolism systemically to potentially mitigate detrimental effects of seed and improve animal performance.

**Keywords:** beef cattle; endophyte; ergot alkaloid; fescue toxicosis; isoflavone; metabolites

**Key Contribution:** As fescue toxicosis causes multiple symptoms that negatively impact beef cattle performance, isoflavone consumption may reduce these effects. The current study found tall fescue seed type to mainly impact the rumen metabolome, while isoflavone supplementation affected the host metabolome in the serum, potentially improving animal growth and development during fescue toxicosis.

#### **1. Introduction**

Tall fescue is the major forage used to feed cattle in pasture-based systems of the southeast and covers approximately 14 million hectares across the United States [1]. The advantage of tall fescue is hardiness of the plants attributed to the presence of a fungal endophyte (*Epichloë coenophialum*, formerly known as *Neotyphodium coenophialum* and *Acremonium coenophialum*) living in a mutualistic relationship with the plant [2]. However, the endophyte produces ergot alkaloids that are toxic to animals that consume them for an extended period of time [3]. Ergot alkaloids are able to bind biogenic amine receptors on blood vessels, resulting in vasoconstriction throughout the body [4–6]. This results in a condition known as fescue toxicosis, which is commonly observed by the animal's inability to thermoregulate [7], poor reproductive performance [1], and reduced average daily gain [8], significantly reducing overall animal performance. Therefore, researchers are tasked with identifying management methods and therapeutics to alleviate these consequences to cattle producers.

Pasture management methods have been evaluated for reducing the impact of fescue toxicosis in cattle. Inter-seeding of legumes, such as red clover, to mitigate the effects of fescue toxicosis has proved beneficial in cattle grazing endophyte-infected tall fescue [9]. Recent research has found phytoestrogenic compounds, known as isoflavones, present in red clover may be responsible for reducing the effects of fescue toxicosis. Isoflavones act as an agonist on the β-adrenergic receptors present on blood vessels to promote vasodilation [10], reversing the effects of ergot alkaloid induced vasoconstriction. Additionally, isoflavones act as a natural antibiotic selective against hyper-ammonia-producing bacteria (HAB) and some cellulolytic and amylolytic bacteria [11,12]. The reduction of ammonia levels as a result of less HAB in the rumen allows more amino acids to be absorbed and used by the ruminant, while altered celluloytic and amylolytic bacteria can influence the production of volatile fatty acids for energy. Therefore, the increase in blood flow and altered rumen fermentation may improve nutrient delivery and utilization for host metabolic processes contributing to animal growth.

The objective of the present study is to evaluate the effect of isoflavone supplementation with tall fescue seed consumption on beef steer's rumen and serum metabolomes. Ruminal and circulating metabolites may provide insights into altered bacterial and host metabolic functions that improve steer performance on endophyte infected tall fescue with the administration of isoflavones.

#### **2. Results**

#### *2.1. Global Rumen Fluid and Serum Metabolome Comparison*

An orthogonal partial least squares discriminant analysis (O-PLS-DA) was used to depict the relationship between the global rumen and serum metabolomes, which illustrated distinct separation between the two metabolomes (Figure 1). A heatmap was also used to visualize the top 25 rumen fluid and serum metabolites by individual steer (Figure 2). The heatmap supports that there is very little similarity between the overall ruminal and circulating metabolites.

#### *2.2. Rumen Fluid Metabolome*

All identified rumen fluid metabolites are presented in Supplementary File 1 with means and standard errors of the mean by treatment combination group. To visualize the effect between steers of the E+P+ and E−P− groups on the rumen fluid metabolome, a partial least squares discriminant analysis (PLS-DA) was created and a significant distinct overlap among seed type was noted (Figure 3). Correlation analyses were performed to analyze the correlation of individual rumen metabolites with the treatment combination groups, and variable importance in the projection (VIP) scores were generated to determine the metabolites that contributed to variation in rumen fluid metabolomes among treatment combination groups. Xylose was negatively correlated with the treatments (r = −0.57) and had one of the greatest impacts on metabolome differences among all treatments (*p* = 0.01) (Figure 4, Table 1). Individual metabolite and metabolic pathway analyses were not significantly impacted by the interaction of seed type and isoflavone treatments (*p* > 0.05).

*Toxins* **2020**, *12*, 744

The rumen metabolome was analyzed by the main effects of seed type and isoflavone treatment. In order to visualize the difference in rumen fluid metabolomes, an O-PLS-DA was generated for the main effects of seed type (Figure 5A) and isoflavone treatment (Figure 5B). For seed type and isoflavone treatment, partial separation was observed between endophyte-infected and endophyte-free seed (Figure 5A). Additionally, partial separation was observed between steers receiving isoflavones and those that did not receive isoflavones (Figure 5B). Correlation analysis indicated hypoxanthine was negatively correlated (r = −0.56) and determined by VIP analysis to have a significant impact on the rumen fluid metabolome differences between endophyte-infected and endophyte-free seed treatments (*p* = 0.01; Figure 6A; Table 1). For isoflavone treatments, trehalose/sucrose was positively correlated (r = 0.06), but had no impact on the rumen fluid metabolome differences (Figure 6B). Metabolites that differed by seed type are presented in Table 1. Eleven metabolites differed significantly as a result of endophyte-infected versus endophyte-free treatments (*p* < 0.05, Table 1). No individual metabolite differences were observed in the rumen fluid as a result of isoflavone treatment. Metabolic pathways that differed significantly by seed type or isoflavone treatment are presented in Table 2. Twenty metabolic pathways were affected by seed type, but only two pathways were affected by isoflavones (*p* < 0.05).

**Figure 1.** Orthogonal partial least squares discriminant analysis (O-PLS-DA) visualizing separation of rumen fluid (triangle) and serum (plus-sign) metabolomes. Ellipse represents a 95% confidence interval.

**Figure 2.** Heatmap of top 25 metabolites of rumen fluid and serum metabolomes by individual steers. Rumen fluid is represented by the red square at the top of the heatmap and serum metabolites are represented by the green squares.


**Table 1.** Rumen fluid metabolites that significantly differed by seed type.

\* Analysis based on ranked data; † values are measured as mean ± SEM of area under the peak; € significance determined at *p* ≤ 0.05 based on FDR-corrected *p*-values; AB within-row represent groupings based on Fisher's LSD.

**Figure 3.** Partial least squares discriminant analysis (PLS-DA) visualizing differences in rumen fluid metabolomes between endophyte-free seed without isoflavones (triangle), endophyte-free with isoflavones (plus-sign), endophyte-infected without isoflavones (multiplication-sign), and endophyte-infected with isoflavones (diamond) treatment groups. Ellipse represents a 95% confidence interval.




**Figure 4.** Variable importance in the projection (VIP) plot indicates xylose to have the greatest influence on the differences in rumen fluid metabolomes between all treatment groups.

**−**

≤

−

− − **Figure 5.** Orthogonal partial least squares discriminant analyses (O-PLS-DA) visualizing differences in rumen fluid metabolomes by seed type (**A**) and isoflavone (**B**) treatments. For seed type (**A**), endophyte-free (E−) steers are represented by a triangle and endophyte-infected (E+) steers by a plus-sign. For isoflavone treatments (**B**), steers receiving isoflavones (P+) are represented by a plus-sign and without isoflavones (P−) by a triangle. Ellipse represents a 95% confidence interval.

− − **Figure 6.** Variable importance in the projection (VIP) plots indicate hypoxanthine to have the greatest influence on the differences in rumen fluid metabolomes between endophyte-free (E−) and endophyte-infected (E+) seed treatment groups (**A**), and trehalose sucrose to have the greatest influence between isoflavone treated (P+) and control (P−) groups (**B**).

#### *2.3. Serum Metabolome*

− − − − − − − − All identified serum metabolites are presented in Supplementary File 2 with means and standard errors of the mean by treatment combination group. The serum metabolome was first analyzed by treatment combination group, isoflavone × seed type. The PLS-DA analysis indicated significant overlap among groups, with partial separation between the E+P+ and E−P− groups (Figure 7). Correlation analyses were performed to determine the correlation of individual serum metabolites with the treatment combination groups and VIP were generated to determine the metabolites that contributed to variation in serum metabolomes among treatment combination groups. Pantothenate

− − − − − − − − − − − − − −

− −

− − − was negatively correlated with interaction of seed type × isoflavone treatments (r = −0.29) and had the largest impact on metabolome differences, although not significant (*p* = 0.07; Figure 8). −

− −

**Figure 7.** Partial least squares discriminant analysis (PLS-DA) visualizing differences in serum metabolomes between all treatment groups: endophyte-free seed without isoflavones (triangle), endophyte-free with isoflavones (plus-sign), endophyte-infected without isoflavones (multiplication-sign), and endophyte-infected with isoflavones (diamond). Ellipse represents a 95% confidence interval.

Similar to rumen fluid, no individual serum metabolites or metabolic pathways were affected by the interaction of seed type and isoflavone treatment (*p* > 0.05). The serum metabolome was then analyzed by the main effects of seed type or isoflavone treatment. In order to visualize the difference in serum metabolomes, O-PLS-DA analyses were generated for seed type (Figure 9A) and isoflavone treatment (Figure 9B). For seed type, partial separation was observed between E+ and E− seed groups (Figure 9A). However, complete separation of serum metabolomes was illustrated between steers receiving isoflavones and those that did not receive isoflavones (Figure 9B). Correlation analysis indicated AMP was negatively correlated with seed treatment (r = −0.35) and determined by VIP analysis to have the greatest impact on serum metabolome differences between E+ and E− steers (*p* = 0.03; Figure 10A). Between isoflavone treatment groups, citrulline was positively correlated (r = 0.47) and had the greatest impact on serum metabolome differences (*p* = 0.003; Figure 10B). Seven metabolites differed significantly as a result of isoflavone treatment (*p* < 0.05, Table 3), while no metabolites differed as a result of seed type (*p* > 0.05). Thirteen metabolic pathways differed (*p* < 0.05) as a result of seed type including glyoxylate and dicarboxylate metabolism; arginine biosynthesis; and alanine, aspartate, and glutamate metabolism (*p* < 0.01; Table 4). For isoflavone treatments, eight metabolic pathways were affected (*p* < 0.05), including pyrimidine metabolism and arginine and proline metabolism (*p* < 0.01; Table 4).

208

**Figure 8.** Variable importance in the projection (VIP) plot indicates pantothenate to have the greatest influence on the differences in serum metabolomes between all treatment groups.

− − **Figure 9.** Orthogonal partial least squares discriminant analyses (O-PLS-DA) visualizing differences in serum metabolomes by seed type (**A**) and isoflavone (**B**) treatments. For seed type (**A**), endophyte-free (E−) steers are represented by a triangle and endophyte-infected (E+) steers by a plus-sign. For isoflavone treatments (**B**), steers receiving isoflavones (P+) are represented by a plus-sign and without isoflavones (P−) by a triangle. Ellipse represents a 95% confidence interval.

−

−

−

−

− − **Figure 10.** Variable importance in the projection (VIP) plot indicates AMP to have the greatest influence on the differences in serum metabolomes between endophyte-free (E−) and endophyte-infected (E+) seed treatment groups (**A**), and citrulline to have the greatest influence between isoflavone treated (P+) and control (P−) groups (**B**).


**Table 3.** Individual serum metabolites that significantly differed by isoflavone treatment.

\* Analysis based on ranked data; † values are measured as mean ± SEM of area under the peak; € significance determined at *p* ≤ 0.05 based on FDR-corrected *p*-values; AB within-row represent groupings based on Fisher's LSD.



**Table 4.** *Cont.*

#### **3. Discussion**

The overall reductions in animal performance due to fescue toxicosis are estimated to cost the cattle industry over \$2 billion annually [13,14]. Therefore, it is vital to discover management methods to reduce the impact and improve the efficiency of beef production. The objective of the current study was to use untargeted metabolomics to evaluate tall fescue seed and isoflavone consumption effects on metabolic intermediates, outputs, and pathways in the rumen and serum.

The metabolomes of the rumen and circulatory environments were first compared, independent of treatment groups, which observed distinctly unique metabolomes according to principal coordinate and abundance analyses. Highly abundant metabolites in each environment were not shared or only present in low abundances between the two environments. The metabolites identified between these different body systems are likely a result of the specific physiological functions of each system in the ruminant. The microbiome is a major contributor to rumen metabolome, as it supplies over 70% of the ruminant's required nutrients [15]. These microbes are highly metabolically active in order to break down feedstuffs and release metabolites to complement host metabolism of which metabolites originate from the plants and other feedstuffs consumed [16]. Therefore, the majority of metabolites identified in the rumen are of xenobiotic origin. By evaluating the rumen metabolome, the effects of tall fescue seed and isoflavone consumption on rumen microbial metabolic processes can be inferred. The serum metabolites, however, are a result of absorbed metabolic products from the rumen and other organs. Tissues throughout the body produce intermediary metabolites from protein, carbohydrate, and lipid metabolism for energy production to perform physiological functions. These metabolites are then absorbed into the blood and can travel through the circulatory system to other tissues for further catabolic or anabolic processing. Therefore, the metabolome of the circulatory system is typically dominated by endogenous metabolites. Because of this systemic nature, metabolites in blood have been used as potential biomarkers to predict feed utilization [17] and production parameters [18], as well as evaluate responses to disease [19,20] and stress [21]. Evaluating the serum metabolome will determine the systemic metabolic response to tall fescue seed and isoflavone supplementation. Together, the effects on individual metabolites and metabolic pathways in rumen fluid and serum will determine how alterations in microbial and host metabolism contribute to symptoms of fescue toxicosis or the benefits isoflavones may contribute to mitigate these detrimental impacts.

Reductions in average daily gain and delayed development of beef cattle are a major consequence of fescue toxicosis [8,22]. The rumen microbiome is crucial for providing nutrients needed by the host for energy requirements and muscle development. As the microbiome has previously been shown to be affected by consuming endophyte infected tall fescue [12,23], the metabolites and other products produced by the rumen microorganisms may be altered, potentially contributing to reductions in growth and feed efficiency. The metabolites produced by the rumen microorganisms are a result of the richness of the rumen microbiome. Several of these metabolites released may be related to the consumption of ergot alkaloids concentrated on the endophyte infected tall fescue seed. Many of the rumen metabolites have a relationship with purine, carbohydrate, and nucleic acid metabolism, such as hypoxanthine, xylose, and uracil, respectively [24,25]; these metabolites are related to feed efficiency parameters. Clemmons et al. [26] found that these metabolites are bio-indicators of feed efficiency in cattle showing low residual feed intake. Interestingly, they are negatively correlated to seed type in the rumen fluid of the current study; it is evident that animals are being affected by the detrimental symptoms of tall fescue toxicosis, failing to gain weight, and being less feed-efficient. Additionally, we did not observe a large number of different metabolites because of the reduction of the rumen microbiome. As a normal rumen environment, the significant presence of other metabolites, which are crucial for the ergot alkaloids metabolism and production of volatile fatty acids, was expected and improves feed efficiency and rumen microbial richness.

Vasoconstriction, induced by ergot alkaloids, occurs throughout the body, resulting in multiple observed symptoms of fescue toxicosis [4,6]. Specifically, the contractility of the mesenteric vasculature surrounding the digestive tract is affected by the consumption of ergot alkaloids, potentially affecting nutrient absorption and subsequent host metabolism [5]. However, the consumption of isoflavones promotes vasodilation to increase blood flow and mitigate fescue toxicosis effects [10]. Ideally, oxygen and nutrient delivery to tissues is improved, thus benefiting host metabolism. Evaluating serum metabolites during induced fescue toxicosis and treatment with isoflavones may indicate the changes in metabolism systemically. The serum metabolome in the current study was greatly affected by isoflavone treatments with complete separation of animals' global serum metabolomes between treatment groups; no metabolites differed as a result of seed type. Citrulline was identified as having the greatest influence on serum metabolomes between isoflavone treatment groups. Citrulline is an intermediary metabolite in the urea cycle, a metabolic process crucial for providing non-protein nitrogen to the ruminant [27]. As isoflavones inhibit hyper-ammonia producing bacteria in the rumen, this reduces the amount of protein degradation, leading to decreased ammonia and nitrogen availability [11,28]. Multiple metabolic pathways related to the urea cycle such as arginine biosynthesis and metabolism, pyrimidine metabolism, and nitrogen metabolism were affected in serum metabolites by isoflavone treatments. Therefore, the effect of citrulline on the serum metabolome due to isoflavone treatment may be a result of changes in the urea cycle by improving protein availability to the ruminant for muscle development.

Pantothenate was considered to be a major contributor to differences observed in the global serum metabolome among all treatment combinations, but was significantly higher in abundance in steers receiving isoflavone treatment. Previous studies have indicated different levels of pantothenate in the serum of animals differing in feed efficiency, with more feed efficient animals having greater serum levels of pantothenate [17,26]. Pantothenate is a key intermediary metabolite for the formation of Coenzyme A, which is crucial for amino acid and lipid metabolism for ruminant muscle development [29]. Additionally, the majority of metabolic pathways affected by isoflavone treatment in the current study were related to amino acid metabolism and biosynthesis. As animals experiencing fescue toxicosis often have low average daily gains [8], the use of isoflavone supplementation may mitigate the weight gain and growth consequences of fescue toxicosis. The greater amount of available pantothenate in the serum of animals consuming isoflavones, with changes in the urea cycling of the ruminant, may improve growth and muscle development in steers affected by fescue toxicosis.

A classic symptom of fescue toxicosis is a significant reduction of prolactin secretion; this is due to the similar homology of ergot alkaloids with the neurotransmitter dopamine. Ergot alkaloids will act as an agonist by binding dopamine receptors, preventing the release of prolactin [3,30]. Tyrosine is a precursor for the generation of the neurotransmitter dopamine [31]. The current study found tyrosine metabolism to be affected by seed type in the rumen; tyrosine metabolic pathways are influenced as the signals for dopamine production are reduced during fescue toxicosis. Additionally, the study found tryptophan metabolism was affected by seed type in the serum. Tryptophan is a key amino acid in regulating protein synthesis, specifically in muscle development, of multiple species [32,33]. The effects of tryptophan on muscle development are through the IGF-1 pathway [33]. Tryptophan is a precursor to the neurotransmitter serotonin, which stimulates the production of

IGF-1 [34]. Supplementation of rumen protected tryptophan improved weight gain and feed efficiency of ruminants [35,36]. The observed impact of seed type on tryptophan metabolism in the serum may indicate reduced production of serotonin and subsequent IGF-1 signaling for muscle development. Together, the impacts of fescue toxicosis on tyrosine and tryptophan metabolism were also observed previously in the plasma of steers consuming endophyte-infected or endophyte-free seed [37,38]. Therefore, tall fescue seed consumption alters neurotransmitter development, leading to commonly observed symptoms of fescue toxicosis.

#### **4. Conclusions**

In conclusion, the rumen metabolome was largely impacted by seed type, while the serum metabolome was influenced by isoflavone supplementation. In the rumen, the impact of the seed type involved carbohydrate and nucleic acids metabolism products of the fescue seed diet inclusion. In the serum, differences in global metabolomes and individual metabolites involved in urea cycling and amino acid metabolic pathways were identified in animals receiving isoflavones and those that did not. Although the low dose of isoflavones administered to cattle indicated effects on the serum and rumen metabolome, further research is needed to determine the effects at other doses. Future applications may lead to the dietary inclusion of isoflavones to reduce the harmful effects of tall fescue toxicosis.

#### **5. Materials and Methods**

All experimental procedures involving animals were approved by the University of Tennessee Institutional Animal Care and Use Committee. The ethic approval code (IACUC) was 2540-0617 and was approved on 20 June 2017.

#### *5.1. Experimental Design and Sample Collection*

Experimental design, animal treatments, and sample collection methods have been previously described in Melchior et al. [12]. Briefly, this study used 36 purebred Angus steers of approximately eight months of age weighing 250 ± 20 kg from Ames Plantation in Grand Junction, TN. Steers were transported to the Plateau Research and Education Center (PREC) in Crossville, TN for the trial, as previously described [12]. Steers were allowed a 10 d acclimation period to the diet formulated to provide 11.57% crude protein and 76.93% total digestible nutrients (DM basis). The GrowSafe System© (GrowSafe Systems Ltd., Calgary, AB, Canada) was used to monitor feed intake. Prior to the beginning of the trial, steers were genotyped for the DRD2 receptor gene, which can influence cattle's response to fescue toxicity [12]. Using this information, the study was blocked on DRD2 genotype, implementing a randomized complete block design. A 2 × 2 factorial arrangement of treatments was utilized with two types of tall fescue including endophyte-infected (E+) and endophyte-free fescue (E−), and treatment with Promensil© (P+) or without (P−) to provide red clover isoflavones. This combination of treatments resulted in four treatment groups: (1) endophyte-infected seed without Promensil (E+P−), (2) endophyte-infected with Promensil (E+P+), (3) endophyte-free without Promensil (E−P−), and (4) endophyte-free with Promensil (E−P+). Within each genotype block, steers were randomly assigned to treatments with nine steers per treatment group. The feed trial occurred over 21 days. In order to provide a consistent amount of ergot alkaloids, endophyte-infected tall fescue seed heads were incorporated into feed to provide a minimum of 0.011 mg ergovaline plus ergovalinine × kg of body weight−<sup>1</sup> (BW) per day [3]. Seed heads were ground through a 5 mm screen using a Wiley Mill (Thomas Scientific, Swedesboro, NJ, USA) and included in feed. A total of 943 mg of isoflavones was provided daily before morning feeding based on previously established dosages [10] using a 28.4 g bolus (Torpac, Inc., Fairfield, NJ, USA) to provide 24.7 g of Promensil. Melchior et al. [12] previously reported the analysis and information of the components present in Promensil, and steers' response to endophyte-infected seed and altered performance parameters. On the final day of the trial (day 21), approximately 9 mL of blood was collected from the coccygeal vein using a serum separator tube (Corvac, Sherwood Medical., St. Louis, MO, USA), and approximately 100 mL of rumen content

was collected via oro-gastric lavage. Blood samples were centrifuged at 2000× *g* and 4 ◦C for 20 min, and serum was transferred to 2 mL microvials and stored at −80 ◦C until metabolite extractions. Rumen samples were centrifuged at 6000× *g* at 4 ◦C for 20 min. The supernatant was aspirated and filtered through a 0.22 µm syringe filter, transferred to 2 mL microvials, and stored at −80 ◦C until metabolite extraction.

#### *5.2. Metabolite Extraction and Identification*

Metabolites were extracted and analyzed as previously described [17] at the UTK Biological and Small Molecule Mass Spectrometry Core (BSMMSC). Briefly, 50 µL of filtered rumen fluid and 50 µL of serum from each steer were extracted using 0.1% formic acid in acetonitrile/water/methanol (2:2:1) using a previously described method [39]. Mobile phases consisted of A: 97:3 water/methanol with 11 mM tributylamine and 15 mM acetic acid and B: methanol, and a gradient consisting of the following: 0.0 min, 0% B; 2.5 min 0% B; 5.0 min, 20% B; 7.5 min, 20% B; 13 min, 55% B; 15.5 min, 95% B; 18.5 min, 95% B; 19 min, 0% B; and 25 min, 0% B; Synergy Hydro-RP column (100 × 2 mm, 2.5 µm particle size) was used to separate metabolites. The flow rate was set to a constant 200 µL/min and the column temperature was kept at 25 ◦C. A Dionex UltiMate 3000 UPLC system (Thermo Fisher Scientific, Waltham, MA) with an autosampler tray maintained at 4 ◦C was used to introduce a 10 µL sample to an Exactive Plus Orbitrap MS (Thermo Fisher Scientific, Waltham, MA, USA) using negative electrospray ionization (ESI) with a capillary temperature of 300 ◦C; spray voltage of 3 kV; and nitrogen sheath and sweep gas at 25 and 3 units, respectively. Data acquisition was done in negative ion mode with a full-scan covering the range of 72–1000 *m*/*z* at 140,000 resolution with automatic gain control of 3 × 10<sup>6</sup> ions [40]. Metabolites are annotated using exact mass of the [M−H]− (±5 pmm) ion and known retention times (±0.3 min) generated from an in-house curated database. The database was created from the analysis of authentic standards and consisted of 300 compounds across various metabolic pathways, focusing on water soluble metabolites in pathways conserved among a diverse array of organisms.

#### *5.3. Metabolite Identification*

Data were analyzed similarly to those of Clemmons et al. [17]. The Xcalibur MS software (Thermo Electron Corp., Waltham, MA) was used to produce raw files, which were then converted to mzML format using ProteoWizard [41]. The software package Metabolomic Analysis and Visualization Engine for LC–MS Data (MAVEN) [42] was used to identify peaks using converted mzML files. MAVEN identifies metabolites based on non-linear retention time correction and calculates peak areas across samples, using a preliminary mass error of ±20 ppm and a retention time window of 5 min. The UTK BSMMSC used a library of 263 retention time-accurate *m*/*z* pairs taken from MS1 spectra for final metabolite annotations. These are based on expansions of previous work [40] and have been replicated at the UTK BSMMSC. The eluted peak of the annotated metabolite had to be found within 2 min of the expected retention time, and the metabolite mass had to be within ±5 ppm of the expected value to be identified as a known compound. The compound area of each peak was calculated using the Quan Browser function of the Xcalibur MS Software (Thermo Electron Corp., Waltham, MA, USA).

#### *5.4. Data Analysis*

Metabolomic data were analyzed using MetaboAnalyst 4.0 [43] and SAS 9.4 (SAS Institute, Cary, NC, USA). For data analysis in MetaboAnalyst, data were first pre-processed. Metabolite data were filtered using interquartile range, normalized by median, log transformed, and auto scaled prior to analysis in MetaboAnalyst 4.0. First, the rumen and serum metabolomes were collectively compared in order to determine similarity of rumen fluid and serum metabolomes for possible overlap or comparison. The rumen and serum metabolomes were visualized using orthogonal partial least squares discriminant analysis (O-PLS-DA) and partial least squares discriminant analysis (PLS-DA) with 2000 permutations. Model fitting for the O-PLS-DA was assessed using R2Y with prediction

power determined using the Q2 metric. A heatmap was generated with the top 25 metabolites to illustrate differences in serum and rumen fluid metabolomes by steer. Next, rumen fluid and serum metabolomes were analyzed separately by treatment combination (i.e., isoflavone × seed type), isoflavone, and seed type. Within each of these, data were visualized via PCA and O-PLS-DA with 2000 permutations, and metabolomes by individual steers were illustrated using heatmaps. Correlation analyses between the top 25 metabolites and treatment groups or combinations were performed for both rumen fluid and serum metabolomes, as well as variable importance in projections (VIP) of the top 25 metabolites. Finally, pathway analyses were performed to determine metabolic pathways that were significantly impacted in rumen fluid and serum by isoflavone or seed type using a global test with relative-betweenness centrality and a reference pathway of Escherichia coli K-12 MG1655 [44].

Raw data were further analyzed in SAS 9.4 (SAS Institute, Cary, NC, USA), First, data were analyzed for normality using the UNIVARIATE procedure, and were considered normal with a Shapiro–Wilk statistic of ≥0.90 and visual observation of histograms and q-q plots. Data that were normally distributed were analyzed with a mixed model analysis of variance (ANOVA) using the GLIMMIX procedure with fixed effects of seed type, isoflavone treatment, and their interaction with the random effect of genotype × isoflavone × seed type. Metabolites that did not follow a normal distribution were fixed ranked and then analyzed using a mixed model ANOVA using the GLIMMIX procedure with fixed effects of seed type, isoflavone treatment, and their interaction with random effect of genotype × isoflavone × seed type.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6651/12/12/744/s1, Supplementary File 1: Means and standard error of the means of all metabolites in the rumen fluid between treatment groups; Supplementary File 2: Means and standard error of the means of all metabolites in the serum between treatment groups.

**Author Contributions:** Conceptualization, E.A.M.-T. and P.R.M.; Formal analysis, B.A.C.; Funding acquisition, P.R.M.; Investigation, T.B.A.-S. and J.F.C.; Methodology, B.A.C., G.E.B., M.D.F., J.L.K., H.J., J.P.G., K.J.M. and P.R.M.; Project administration, P.R.M.; Resources, E.A.M.-T.; Visualization, T.B.A.-S. and B.A.C.; Writing—original draft, T.B.A.-S., B.A.C., J.F.C., K.J.M. and P.R.M.; Writing—review & editing, T.B.A.-S., E.A.M.-T., B.A.C., J.F.C., J.L.K., K.J.M. and P.R.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the University of Tennessee CVM, Center for Excellence in Livestock and Human Diseases.

**Acknowledgments:** We acknowledge the USDA-NIFA Hatch/Multistate Project W4177-TEN00524-Enhancing the Competitiveness and Value of U.S. Beef. We thank Ames Plantation, UT Plateau Research and Education Center, Rebecca Payton, Lezek Wojakiewicz, and Gloria Gellin (USDA-ARS) for their assistance. We also thank Shawn Campagna and Hector Castro at the UTK Biological and Small Molecule Mass Spectrometry Core (BSMMSC).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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## *Article* **Vasoactive Effects of Acute Ergot Exposure in Sheep**

**Rossalin Yonpiam <sup>1</sup> , Jair Gobbet 1 , Ashok Jadhav 2 , Kaushik Desai 2 , Barry Blakley <sup>3</sup> and Ahmad Al-Dissi 1, \***

	- SK S7N 5E5, Canada; ashok.jadhav@cnl.ca (A.J.); k.desai@usask.ca (K.D.)
	- <sup>3</sup> Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine; University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada; barry.blakley@usask.ca
	- **\*** Correspondence: ahmad.aldissi@usask.ca; Tel.: +1-306-966-7643; Fax: +1-306-966-7439

**Abstract:** Ergotism is a common and increasing problem in Saskatchewan's livestock. Chronic exposure to low concentrations of ergot alkaloids is known to cause severe arterial vasoconstriction and gangrene through the activation of adrenergic and serotonergic receptors on vascular smooth muscles. The acute vascular effects of a single oral dose with high-level exposure to ergot alkaloids remain unknown and are examined in this study. This study had two main objectives; the first was to evaluate the role of α<sup>1</sup> -adrenergic receptors in mediating the acute vasocontractile response after single-dose exposure in sheep. The second was to examine whether terazosin (TE) could abolish the vascular contractile effects of ergot alkaloids. Twelve adult female sheep were randomly placed into control and exposure groups (*n* = 6/group). Ergot sclerotia were collected and finely ground. The concentrations of six ergot alkaloids (ergocornine, ergocristine, ergocryptine, ergometrine, ergosine, and ergotamine) were determined using HPLC/MS at Prairie Diagnostic Services Inc., (Saskatoon, SK, Canada). Each ewe within the treatment group received a single oral treatment of ground ergot sclerotia at a dose of 600 µg/kg BW (total ergot) while each ewe in the control group received water. Animals were euthanized 12 h after the treatment, and the pedal artery (dorsal metatarsal III artery) from the left hind limb from each animal was carefully dissected and mounted in an isolated tissue bath. The vascular contractile response to phenylephrine (PE) (α<sup>1</sup> -adrenergic agonist) was compared between the two groups before and after TE (α<sup>1</sup> -adrenergic antagonist) treatment. Acute exposure to ergot alkaloids resulted in a 38% increase in vascular sensitivity to PE compared to control (Ctl EC<sup>50</sup> = 1.74 × 10 <sup>−</sup><sup>6</sup> M; Exp EC<sup>50</sup> = 1.079 × 10 <sup>−</sup><sup>6</sup> M, *p* = 0.046). TE treatment resulted in a significant dose-dependent increase in EC<sup>50</sup> in both exposure and control groups (*p* < 0.05 for all treatments). Surprisingly, TE effect was significantly more pronounced in the ergot exposed group compared to the control group at two of the three concentrations of TE (TE 30 nM, *p* = 0.36; TE 100 nM, *p* < 0.001; TE 300 nM, *p* < 0.001). Similar to chronic exposure, acute exposure to ergot alkaloids results in increased vascular sensitivity to PE. TE is a more potent dose-dependent antagonist for the PE contractile response in sheep exposed to ergot compared to the control group. This study may indicate that the dry gangrene seen in sheep, and likely other species, might be related to the activation of α<sup>1</sup> adrenergic receptor. This effect may be reversed using TE, especially at early stages of the disease before cell death occurs. This study may also indicate that acute-single dose exposure scenario may be useful in the study of vascular effects of ergot alkaloids.

**Keywords:** acute ergot exposure; ergot toxicity; sheep; vasoconstriction; adrenergic receptors

**Key Contribution:** This study demonstrated that, similar to chronic exposure, an acute single-dose oral exposure of sheep to ergot alkaloids results in increased pedal artery sensitivity to phenylephrine.

**Citation:** Yonpiam, R.; Gobbet, J.; Jadhav, A.; Desai, K.; Blakley, B.; Al-Dissi, A. Vasoactive Effects of Acute Ergot Exposure in Sheep. *Toxins* **2021**, *13*, 291. https:// doi.org/10.3390/toxins13040291

Received: 14 February 2021 Accepted: 2 April 2021 Published: 20 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Ergot poisoning remains an economically important disease affecting a variety of animal species including cattle, sheep, horses, and goats with estimated annual losses of more than a billion dollars within the US [1]. Ergot poisoning is caused by the prolonged consumption of ergot alkaloids which are naturally occurring mycotoxins produced by fungi infecting crops such as triticale, cereals, and grains such as barley, wheat, and durum [2–4]. The most widely encountered species of ergot alkaloid producing fungi in Western Canada and Europe are in the family of *Clavicipitaceae* [5–7]. This fungal family includes the external spore-producing fungi (*Claviceps* spp.) and endophytic fungi (*Epichloë* spp.). The major species causing agricultural problems in Western Canada is *Claviceps purpura* [5,6]. The active ingredients of ergot alkaloids are confined and concentrated within the sclerotia which are external fungal bodies [8]. Clinical signs of lameness, hoof loss, and dry gangrene of the lower limbs, tail, ear tips, and teats are commonly seen in chronic ergotism and are related to the effect of ergot alkaloids on the vasculature causing severe vasoconstriction [9–11]. A complete discussion of the impact of ergot alkaloids on various organs or systems is beyond the scope of this paper. Table 1 provides a brief summary of the main effects reported in the literature (reviewed by Strickland J et al., 2011).

**Table 1.** Summary of the main reported effects of ergot alkaloids on different organ or systems in animals (reviewed by Strickland J et al., 2011).


The precise in vivo vasoactive mechanisms of ergot alkaloids have not been determined. However, in vitro tissue bath studies, where normal dissected and isolated arterial rings were exposed to purified ergot alkaloids, have previously shown that the adrenergic and serotonergic receptors on vascular smooth muscles are activated. This is also supported by the fact that the chemical structure, i.e., the ergoline ring, of ergot alkaloids resembles

that of physiologic neurotransmitters such as dopamine, norepinephrine, epinephrine, and serotonin, which are known to be vasoactive [4].

It is important to note that despite the rapid metabolism and excretion of ergot alkaloids which occurs within several hours after exposure [12,13], the clinical vascular manifestations of ergot alkaloids are always seen after the prolonged (several weeks to months) consumption of ergot-contaminated plants. While these clinical vascular manifestations could be explained by the repeated exposure to ergot alkaloids, these effects often remain long after the ergot-contaminated feed is removed! Recent evidence suggests that ergot alkaloids may bioaccumulate within the vasculature [14,15]. It is also possible that other unknown vasoactive mechanisms may be involved in mediating these effects.

It is unknown whether acute ergot exposure affects vascular contractility in a similar manner to chronic exposure. If similar effects are found, then acute exposure scenarios may be useful to study the mechanisms of vascular alteration by ergot alkaloids. Many studies have focused on finding an antagonist to counteract the clinical effects of ergotism. Elucidating the mechanisms of vascular contractile response induced by ergot alkaloids may prove useful to identify treatment options for the vascular-related clinical manifestations of ergot poisoning.

This study aimed to examine the role of adrenergic receptors in mediating the vascular effects of ergot alkaloids after an in vivo acute exposure scenario to these alkaloids. Vascular sensitivity to phenylephrine (PE), an α1-adrenergic agonist, was compared between ergot exposed and control groups before and after terazosin (TE), α1–adrenergic antagonist, treatment.

We hypothesized that an acute single dose oral exposure to ergot alkaloids results in increased vascular sensitivity (decreased EC50) to PE in the pedal artery; an effect that is mediated through the activation of α1-adrenergic receptors. We also hypothesized that the acute vascular effects of oral exposure to ergot alkaloids can be reversed via TE (the α1-adrenergic antagonist).

#### **2. Results**

All animals remained healthy after treatment and did not exhibit any clinical signs during the 12 h period between the administration of ergot alkaloids and euthanasia. No gross or histological changes were seen in either group in the lung, liver, kidneys, heart, spleen, intestines, fat, and pedal arteries. The concentration of ergot alkaloids used to formulate the single oral dose is shown in Table 2.

**Table 2.** The concentration of six ergot alkaloids determined within ground sclerotia using HPLC/MS \*. The total concentration of these alkaloids was used to formulate a single oral dose (600 µg/kg BW) which was administered to each sheep using a stomach tube.


\* The detection limit for each alkaloid was 1.25 ppb. HPLC/MS, high performance liquid chromatography and mass spectrometry; µg/kg BW, microgram per kilogram body weight; ppb, part per billion.

#### *2.1. Phenylephrine Dose Response Curve Compared between Ergot Exposure and Control Groups*

In the control group, the PE contractile response was first observed at 1 × 10−<sup>7</sup> M concentration, and the maximum contractile response recorded at the highest PE concentration

(1 × 10−<sup>4</sup> M) was 22.8 g. The contractile response in the exposure group was first observed at 0.5 × 10−<sup>7</sup> M while the highest PE concentration yielded a maximum contraction of 18.0 g. Ergot exposure resulted in a significant decrease in EC<sup>50</sup> compared to the control group (*p* = 0.0462). Comparisons of EC<sup>50</sup> between the two groups are presented in Figure 1. Details of EC<sup>50</sup> are for all groups are presented in Table 3. − −

−

− **Figure 1.** Mean arterial contractile responses to increasing concentration of PE compared between control and ergot exposed group. The pedal artery was collected 12 h after single oral exposure to 600 µg/kg BW (total ergot) or after placebo water treatment (*n* = 6/group). Contractile response data were presented as percentage means ± *SEM* of the maximum contractile effect induced by 1 × 10−<sup>4</sup> M PE treatment. Ergot exposure resulted in a significant decrease in EC<sup>50</sup> compared to the control group (*p* < 0.05). EC50, the concentration of phenylephrine producing 50% of the maximum contractile response; PE, phenylephrine; BW, body weight; CTL, control group; EXP, exposure group; M, Molar.

**Table 3.** Phenylephrine (PE) EC<sup>50</sup> compared between ergot exposed and control sheep (*n* = 6/group) before and after terazosin treatment in dissected pedal arteries using an arterial tissue bath. Ergot exposed sheep received a single oral dose of 600 µg/kg BW total ergot dissolved in a water based on the levels of six ergot alkaloids determined previously. Control sheep received a water placebo treatment. The effect of terazosin was determined using three increasing concentrations of terazosin: 30, 100, and 300 nM. For each treatment type, a sigmoidal dose-response curve was plotted using nonlinear regression which was used to calculate EC50. Statistical differences in EC<sup>50</sup> among the different treatment types were calculated by the extra sum-of-squares *F*-test. A *p*-value less than 0.05 was considered significant.


a–i letters with the same superscript are significantly different. EC50, the concentration of phenylephrine producing 50% of the maximum contractile response; µg/kg BW, microgram per kilogram body weight; PE, phenylephrine; TE, Terazosin; CI, confidence interval; nM, nanomolar.

#### *2.2. Effect of Terazosin Treatment on Phenylephrine Dose Response Curve*

In the control group, TE treatment resulted in a significant and dose-dependent increase in EC<sup>50</sup> (*p* < 0.0001 for all concentrations; 30, 100, and 300 nM). Similarly, EC<sup>50</sup> significantly increased in a dose-dependent manner in the exposure group after terazosin treatment (*p* < 0.0001 for all concentration; 30, 100, and 300 nM) (Figures 2 and 3). The blocking effect of TE was greater in the exposure group when compared to the control group when given at 100 nM and 300 nM (*p* < 0.0001). A similar trend of increasing EC<sup>50</sup> in the exposure group compared to the control group after the 30 nM TE treatment was seen, but the difference was not statistically significant (*p* = 0.076) (Figures 4–6). (See Table 3 for details).

− **Figure 2.** Mean arterial contractile responses to increasing concentration of PE in control animals compared before and after 30, 100, or 300 nM TE treatment (*n* = 6). Contractile response data were presented as percentage means ± *SEM* of the maximum contractile effect induced by 1 × 10−<sup>4</sup> M PE treatment. TE treatment resulted in a significant dose-dependent increase in EC<sup>50</sup> compared to PE alone (*p* < 0.0001). EC50, the concentration of phenylephrine producing 50% of the maximum contractile response; PE, phenylephrine; TE, terazosin; M, Molar; nM, nanomolar. −

− − **Figure 3.** Mean arterial contractile responses to increasing concentration of PE in ergot exposed animals compared before and after 30, 100, or 300 nM TE treatment (*n* = 6). Contractile response data were presented as percentage means ± *SEM* of the maximum contractile effect induced by 1 × 10−<sup>4</sup> M PE treatment. TE treatment resulted in a significant dose-dependent increase in EC<sup>50</sup> compared to PE alone (*p* < 0.0001). EC50, the concentration of phenylephrine producing 50% of the maximum contractile response; PE, phenylephrine; TE, terazosin; Exp, exposure group; M, Molar; nM, nanomolar.

− **Figure 4.** Mean arterial contractile responses to increasing concentration of PE compared between control and ergot exposed groups after TE treatment at 30 nM. EC<sup>50</sup> was not significantly different between the two groups (*p* = 0.37). Contractile response data were presented as percentage means ± *SEM* of the maximum contractile effect induced by 1 × 10−<sup>4</sup> M PE treatment. EC50, the concentration of phenylephrine producing 50% of the maximum contractile response; PE, phenylephrine; TE, terazosin; CTL, control group; EXP, exposure group; M, Molar; nM, nanomolar. −

− − **Figure 5.** Mean arterial contractile responses to increasing concentration of PE compared between control and exposure after TE treatment at 100 nM. EC<sup>50</sup> was significantly different between the two groups (*p* < 0.0001). Contractile response data were presented as percentage means ± *SEM* of the maximum contractile effect induced by 1 × 10−<sup>4</sup> M PE treatment. EC50, the concentration of phenylephrine producing 50% of the maximum contractile response; PE, phenylephrine; TE, terazosin; CTL, control group; EXP, exposure group; M, Molar; nM, nanomolar.

− **Figure 6.** Mean arterial contractile responses to increasing concentration of PE compared between control and exposure after TE treatment at 300 nM. EC<sup>50</sup> was significantly different between the two groups (*p* < 0.0001). Contractile response data were presented as percentage means ± *SEM* of the maximum contractile effect induced by 1 × 10−<sup>4</sup> M PE treatment. EC50, the concentration of phenylephrine producing 50% of the maximum contractile response; PE, phenylephrine; TE, terazosin; CTL, control group; EXP, exposure group; M, Molar; nM, nanomolar.

#### **3. Discussion**

The biological effects of ergot alkaloids on livestock are known to be diverse. This diversity is not only related to differences in alkaloid concentration and specific alkaloid content in different plants, but also due to their ability to affect multiple biological processes [4,7,16,17]. The ergoline ring system, which is a structure common to ergot alkaloids, is similar to the ring structure of epinephrine, dopamine, and serotonin thus allowing ergot alkaloids to mimic their function. In the vasculature, ergot alkaloids bind with a variety of serotonergic and adrenergic receptors to modify vascular tone [11]. The effects of these alkaloids are known to be diverse with differing potencies among different animal species; however, within a species, the effects are dependent on the animal's general health, body condition, reproductive status and previous exposure [4,18].

α Ergotism in livestock is known to cause dry gangrene due to severe vasoconstriction within peripheral vasculature. Ergotism occurs after the prolonged ingestion of ergot alkaloids. Therefore, previous studies have focused on examining the mechanisms of vasoconstriction following chronic exposure scenarios [19–21]. Thus, the vascular effects following acute exposure remain unknown. In this study, we wanted to investigate the role of α1-adrenergic receptor activation on vascular contractile response following a single acute high-dose of oral exposure scenario to ergot alkaloids using sheep as a model. Similar to other livestock species, sheep are chronically affected by ergotism and develop dry gangrene after prolonged exposure. We chose to examine the pedal artery due to its peripheral location on the ovine limb.

Ideally, pure individual ergot alkaloids should be used in prolonged feeding trials to precisely examine their vascular effects and the mechanism of these effects. However, because pure ergot alkaloids are very expensive, previous studies often used ergot or endophyte-infected tall fescue. It is often difficult to accurately estimate the individual dose in these studies as the concentration of alkaloids within feed is subject to significant variability due to feed storage conditions and uneven distribution. Alternatively, pure individual alkaloids are often used on dissected arteries to examine their vascular contractile effects in vitro using arterial tissue bath systems. In order to achieve a more defined

dosing protocol, we used ground sclerotia in which the concentration of six different ergot alkaloids was determined [16,17]. The dose was adjusted in every animal depending on the body weight to receive a dose of 600 µg/kg BW of total ergot alkaloid content. To the author's knowledge, no studies have been performed to examine the vascular effects of acute single-dose oral exposure to ergot alkaloids. All previous studies reported vascular effects after repeated low-dose exposure. It is likely that a single low-dose oral exposure will not have a detectable impact on the vasculature. Therefore, to increase the odds of detecting vascular contractile effects after single-dose oral exposure, this study opted to use a high-dose of ergot alkaloids. While the repeated exposure to such a high level is unlikely to occur due to feed refusal, pelleted feed submitted to our diagnostic laboratory from livestock producers for ergot testing occasionally contained similarly high levels.

It is known that the degree of vasoconstriction induced by ergot alkaloids is alkaloid dependent. For example, the vasoconstrictive effect elicited by ergocryptine is 100 times less potent as compared to ergotamine, whereas ergocristine and ergocornine are only 10 times less potent. Ergovaline, the predominant alkaloid in tall fescue grass, is thought to have a similar potency to ergotamine [11,22]. The potency of these alkaloids varies depending on their relative binding affinity to α-adrenergic and serotonergic receptors and their ability to specifically activate them. Most studies examining the vascular effects of ergot alkaloids have focused on studying the serotonergic receptors [19,23–25]. However, very few studies examined the activation of α-adrenergic receptors by different alkaloids. For example, the contractile response in the lateral saphenous vein of cattle grazing tall fescue was significantly enhanced compared to control animals by BHT-920, an α2-adrenergic agonist, but not by PE (α1-adrenergic agonist) [26]. In addition, Schöning et al. reported that ergovaline stimulated α1-adrenergic receptors but with low efficacy in rat thoracic aorta [22]. In vivo studies focusing on heart rate and blood pressure changes after exposure to ergot alkaloids also indicate α-adrenergic receptor activation. Bradycardia induced by ergotamine in anesthetized rats was reduced by yohimbine, an α2-adrenergic antagonist. In addition, ergotamine treatment reduced the tachycardia induced by electrical stimulation of the spinal cord, and the reduction was similarly blocked after yohimbine treatment [27]. Similarly, in rats, ergotamine has been shown to act as an agonist on α2-adrenergic receptors and an antagonist on α1-adrenergic receptor [28]. In our study, a significant increase in vascular sensitivity to PE was found in ergot exposed sheep compared to control animals, which might suggest that α1-adrenergic receptors mediate that response.

Similar to what we expected, TE decreased the vascular sensitivity to PE in ergot exposed and control sheep due to its antagonistic effects on the α1-adrenergic receptor. However, surprisingly, the potency of TE as an α1-adrenergic receptor antagonist was significantly enhanced in ergot exposed sheep compared to controls. It has been recently shown that previous exposure to high concentrations of ergot alkaloids may decrease vascular contractility making the vasculature less susceptible to the effects of ergot alkaloids. Klotz et al. examined the contractile response to ergovaline in cattle chronically grazing high and low-endophyte-infected tall fescue [23]. This study demonstrated that the maximum contractile response was significantly higher in steers consuming low-endophyte-infected tall fescue. This is contrary to other studies, which found that the increase in vascular contractile response to ergot alkaloids is dose-dependent [24,29]. It is, thus, possible that the vascular contractile effect of ergot alkaloids is dose-dependent but may become less effective at very high doses. It is possible that the high dose of ergot alkaloids we used resulted in a relatively low contractile response, and also enhanced the blocking effect of TE resulting in a reduced contractile response compared to control tissues. The increased blocking sensitivity of TE in exposed animals may also indicate that the full impact of ergot exposure was not realized due to the short exposure duration.

Alternatively, it is also possible that the effect of the blocker was enhanced in the ergot exposed group due to the unique mixture of alkaloids in the diet. Interestingly, it has been shown that the presence of ergocristine, ergocornine, and ergocryptine together produces adrenergic blockade [30–32]. Additionally, Roquebert and Demichel reported that ergocristine acts as an α1-adrenergic blocker in rat tail artery [28,33,34]. Ergocristine had the highest concentration in the diet used in this study and may have acted as an antagonist. The enhanced blocking effect of TE in ergot exposed animals may indicate that this blocker may be useful in counteracting the vascular effects of ergot alkaloid exposure.

Several studies have shown that ergot alkaloids interact with serotonin receptors in chronic exposure scenario [19,24,25]. However, Kalkman et al. reported that in rats injected intravenously with ergometrine, the vasoconstrictor response was related to the activation of α1- and α2-adrenergic receptors, but not serotonergic receptors [35]. It would be interesting to examine the role of serotonergic receptors in mediating arterial contraction after acute exposure to ergot alkaloids. High-level exposure in ruminants can result in nervous signs such as hyperexcitability, hypermetria, and tremors [11,36]. The dose we used was relatively high but was well tolerated by all animals, with none showing clinical signs of illness.

Currently, ergot toxicity is thought to be only related to the prolonged consumption of ergot alkaloids, and it is presumed that a short-term exposure will have no significant clinical effects. However, we show for the first time that even a single oral dose of ergot alkaloids causes a significant increase in sensitivity in arteries supplying distal extremities. This finding is of significance to the livestock industry and regulators, as it may indicate that in cold weather conditions, short-term exposure to ergot alkaloids may result in a significant decrease in blood supply to the extremities, making animals prone to gangrene. It would be interesting to examine whether a similar but lower level exposure scenario would result in a similar response in livestock. In addition, it is also important to examine the effects of a short-term exposure on other systems as it is now presumed that the effects are only seen after chronic exposure. If similar negative effects are seen in other systems, it may indicate the need to lower the allowable limits of ergot alkaloids within feed to reflect the true nature of the negative impact of this disease.

The finding that a blocking effect of TE was more potent in ergot exposed animals may indicate that this drug could be used to treat animals who have been recently exposed to ergot alkaloids. If it is proved to be useful, this drug may significantly reduce the economic impact of ergotism to the livestock industry. It would be interesting to examine whether TE has any impact on other systems affected by this disease.

We recently showed that the S-epimers of ergot alkaloids are vasoactive causing vasoconstriction of bovine dorsal metatarsal arteries in vitro [37]. It is, therefore, possible that the effects seen in this study are related to the combined activity of the R and S epimers and not just the R-epimers.

In summary, this study found that acute high-level exposure to ergot alkaloids results in increased vascular sensitivity to PE and increased blocking effect of TE. Additional studies are immensely needed to examine the role of adrenergic and, serotonergic receptors in other vascular beds in vivo and in vitro after acute exposure.

#### **4. Materials and Methods**

#### *4.1. Animals*

All protocols were approved by the Animal Care and Ethics Committee at the University of Saskatchewan (Animal Use Protocol # 20150047, approval date: 31 August 2016). Before the experiment, all animals were weighed and clinically examined with body temperature and heart rate recorded. A blood sample was also collected from each animal, and a complete blood count was performed evaluating red and white blood cell counts as well as platelets count and total plasma protein to ensure that all animals were healthy.

#### *4.2. Tissue Collection & Stock Solutions*

Twelve healthy adult ewes were randomly assigned into treatment or control groups (*n* = 6/group). Animals were allowed to acclimatize for fourteen days and were fed alfalfa hay and water ad libitum. Ergot alkaloids containing sclerotia were collected, finely ground and the concentrations of six alkaloids (ergocornine, ergocristine, ergocryptine, ergometrine,

ergosine, and ergotamine) were determined using HPLC/MS at Prairie Diagnostic Services Inc. (PDS), Saskatoon, SK, Canada (16). Each ewe within the treatment group received a single dose of ground ergot sclerotia at a dose of 600 µg/kg BW (total ergot) dissolved in 50 mL of water via a stomach tube. The concentrations of ergot alkaloids within sclerotia are recorded in Table 1. The control group received water placebo. Twelve hours after treatment, animals were euthanized using a captive bolt and a necropsy was performed. A 15 cm segment of the pedal artery (dorsal metatarsal artery III) was carefully dissected and collected from each animal, soaked in a diluted heparin solution (10 Unit/1 mL) for 2 min and transferred into a container containing modified Krebs–Henseleit buffer solution [in mM: 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 22.0 NaHCO3, 5.0 glucose and 2.5 CaCl2; (Sigma-Aldrich Canada Ltd. Oakville, ON, Canada) (pH 7.4 gassed with 95% O2, 5% CO<sup>2</sup> at 37 ◦C)] on ice until transport to the laboratory. Immediately upon arrival to the lab, adipose and connective tissue were carefully removed from each arterial segment, which was later sliced into four 3 to 5 mm cross sections. Each arterial section was suspended between the bases of two triangular-shaped wires within an isolated 10 mL tissue bath (Chengdu equipment manufacturing, China) containing modified Krebs–Henseleit buffer solution maintained at the above conditions. Arterial rings were allowed to equilibrate for 1 h under a resting tension of 2 g with the bath solution changed every 15 min. Each day, a fresh stock solution was prepared for phenylephrine (PE) (Sigma-Aldrich Canada Ltd. Oakville, ON, Canada) at a concentration of 1 M, followed by a 10-fold serial dilution to prepare the remaining working solutions. 10 µL were added from each dilution to the 10 mL incubation buffer to obtain the desired final concentration (1 × 10−9–1 × 10−<sup>4</sup> M). Similarly, a fresh initial 1 M stock solution of terazosin (Sigma-Aldrich Canada Ltd. Oakville, ON, Canada) was prepared from which a 10 µM and 100 µM dilutions were made. The desired final concentrations of 30, 100, and 300 nM were prepared by adding 30 µL from the first stock or 10 and 30 µL from the second stock as appropriate. Arterial rings were treated with PE (1 × 10−<sup>4</sup> M) (Sigma-Aldrich Canada Ltd. Oakville, ON, Canada) to initiate contraction and to confirm tissue viability and responsiveness. The tissues were later washed with incubation buffer until resting tension was achieved.

#### *4.3. Contractile Response*

Three vascular rings from the pedal artery of each animal were used to assess the PE contractile response before and after the incubation of each ring with a different concentration of TE. Initially, a cumulative concentration-dependent contraction in response to PE was obtained by adding increasing concentrations of PE (1 × 10−<sup>9</sup> M to 1 × 10−<sup>4</sup> M). After each PE treatment, arterial rings were allowed to achieve maximum tension which plateaued for 2 min before the next concentration was added. After the last PE treatment, arterial rings were allowed to return to resting tension with buffer replacement occurring every 15 min for 1 h. This was followed by incubating each of the three rings with 30, 100 or 300 nM TE for 20 min after which the cumulative PE contractile response was repeated in each chamber as above. Following completion of the exposure, all rings were exposed to 1 × 10−<sup>4</sup> M PE to verify their viability.

#### *4.4. Data Collection, Analysis and Statistical Analysis*

All measured isometric contractile responses were recorded in grams of tension using 'Chart' software and Powerlab equipment (AD Instruments Inc., Colorado Springs, CO, USA). For each PE treatment, the maximum tension in grams achieved before the 2 min plateau period was recorded and corrected for a baseline. To minimize variation due to arterial size, each contractile response from an individual ring was normalized to its maximum contractile tension induced by 1 × 10−<sup>4</sup> M PE treatment.

Contractile response data were presented as percentage means ± *SEM* of the maximum contractile effect induced by 1 × 10−<sup>4</sup> M PE treatment. For each treatment type, a sigmoidal dose-response curve was plotted using nonlinear regression with variable slope utilizing GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA, USA), which

was later used to calculate potency presented as the concentration producing 50% of the maximum response (EC50). Results were presented as the log of the EC<sup>50</sup> value. Statistical differences in EC<sup>50</sup> among the different dose-response curves were calculated by the extra sum-of-squares *F*-test where a *p*-value less than 0.05 was considered significant.

**Author Contributions:** Formal analysis, R.Y. and A.A.-D.; Funding acquisition, B.B. and A.A.-D.; Investigation, R.Y., J.G., A.J., K.D., and A.A.-D.; Methodology, A.J., K.D., B.B., and A.A.-D.; Project administration, R.Y., B.B., and A.A.-D.; Resources, K.D. and A.A.-D.; Supervision, A.A.-D.; Writing– original draft, R.Y.; Writing–review & editing, K.D., B.B., and A.A.-D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research and the APC were funded by Agriculture Development Fund of Saskatchewan, grant number 20140186.

**Institutional Review Board Statement:** This study was approved by the University of Saskatchewan Animal Research Ethics Board. Animal Use Protocol # 20150047. Approval date: 31 August 2016.

**Informed Consent Statement:** Informed consent was not needed because all studied subjects were animals.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Funding for this project was kindly provided by a reseach grant from the Saskatchewan Agriculture Development Fund recevived by AN. Al-Dissi as the primary investigator.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


#### *Article* **Influence of Prolonged Serotonin and Ergovaline Pre-Exposure on Vasoconstriction Ex Vivo †**

**Eriton E. L. Valente 1 , David L. Harmon <sup>2</sup> and James L. Klotz 3, \***


**Abstract:** Ergot alkaloid mycotoxins interfere in many functions associated with serotonergic neurotransmitters. Therefore, the objective was to evaluate whether the association of serotonin (5 hydroxytryptamine, 5-HT) and ergot alkaloids during a 24 h pre-incubation could affect the vascular contractile response to ergot alkaloids. To evaluate the effects of 24 h exposure to 5-HT and ergot alkaloids (ergovaline, ERV), two assays were conducted. The first assay determined the half-maximal inhibitory concentration (IC<sup>50</sup> ) following the 24 h pre-exposure period, while the second assay evaluated the effect of IC<sup>50</sup> concentrations of 5-HT and ERV either individually or in combination. There was an interaction between previous exposure to 5-HT and ERV. Previous exposure to 5-HT at the IC<sup>50</sup> concentration of 7.57 × 10 <sup>−</sup><sup>7</sup> M reduced the contractile response by more than 50% of control, while the exposure to ERV at IC<sup>50</sup> dose of 1.57 × 10 <sup>−</sup><sup>10</sup> M tended to decrease (*p* = 0.081) vessel contractility with a response higher than 50% of control. The 24 h previous exposure to both 5-HT and ERV did not potentiate the inhibitory response of blood vessels in comparison with incubation with each compound alone. These results suggest receptor competition between 5-HT and ERV. More studies are necessary to determine the potential of 5-HT to treat toxicosis caused by ergot alkaloids.

**Keywords:** blood vessel; ergovaline; myograph; serotonin

**Key Contribution:** The inhibitory effect of prolonged exposure to 5-HT and ergot alkaloids on vascular contractility was demonstrated. The possible competition between 5-HT and ergot alkaloids for serotonergic receptors should be explored as a potential therapy for ergot toxicosis.

#### **1. Introduction**

Ergot alkaloid mycotoxins have a significant impact on livestock health and productivity globally [1]. The similarities between the tetracyclic ergoline ring, common to naturally occurring ergot alkaloids, and the ring structure of the biogenic amine neurotransmitters allows ergot alkaloids, like ergovaline to interfere in the many functions associated with these neurotransmitters [2]. After a period of feeding ergot alkaloids, some serotonergic receptors become less responsive to serotonin (5-HT) [3–5]. Drugs that are derivatives of ergot alkaloids have an association and dissociation rate to serotonin 5-HT<sup>2</sup> receptors that is much slower than 5-HT [6], causing long-term stimulation. This persistent stimulation of serotonergic receptors has been shown to result in blunted signaling [7] and this alters how tissues respond to receptor-driven stimuli. This is exemplified in ergot alkaloid mycotoxicosis by a sustained vasoconstriction that limits effective blood flow to visceral and peripheral tissues [2]. Many symptoms of ergotism, like fescue toxicosis, are related to vasoconstriction, which is associated with lower blood flow [8,9] causing gangrene [10] and

**Citation:** Valente, E.E.L.; Harmon, D.L.; Klotz, J.L. Influence of Prolonged Serotonin and Ergovaline Pre-Exposure on Vasoconstriction Ex Vivo. *Toxins* **2022**, *14*, 9. https:// doi.org/10.3390/toxins14010009

Received: 5 November 2021 Accepted: 18 December 2021 Published: 23 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

affecting thermal regulation [11]. Currently, there is no effective treatment for mycotoxicosis caused by ergot alkaloids in livestock. ing gangrene [10] and affecting thermal regulation [11]. Currently, there is no effective treatment for mycotoxicosis caused by ergot alkaloids in livestock. Agonist therapy is the use of stimulant-like medications to treat stimulant addictions

flow to visceral and peripheral tissues [2]. Many symptoms of ergotism, like fescue toxicosis, are related to vasoconstriction, which is associated with lower blood flow [8,9] caus-

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Agonist therapy is the use of stimulant-like medications to treat stimulant addictions and has proven an effective treatment for dependence on neuromodulator drugs, such as nicotine and opioids [12]. This strategy involves the administration of medications that share neurobiological mechanisms with the undesirable drug in pursuit of neurochemical normalization [13]. The exposure to ergot alkaloids can produce neurochemical deficits in 5-HT [14]. Thus, the ideal agonist therapy would normalize 5-HT dysfunction. and has proven an effective treatment for dependence on neuromodulator drugs, such as nicotine and opioids [12]. This strategy involves the administration of medications that share neurobiological mechanisms with the undesirable drug in pursuit of neurochemical normalization [13]. The exposure to ergot alkaloids can produce neurochemical deficits in 5-HT [14]. Thus, the ideal agonist therapy would normalize 5-HT dysfunction. Traditionally, agonist therapy uses agonists with lower potency [12]. The less persis-

Traditionally, agonist therapy uses agonists with lower potency [12]. The less persistent receptor association with 5-HT in comparison with ergot alkaloid derivatives [6] qualifies 5-HT as a potential molecule for agonist therapy for ergot alkaloid toxicosis. Hypothetically, the increase of 5-HT in the synaptic cleft could intensify competition with ergot alkaloids at the serotonergic receptors and reduce the overstimulation through a reduction in ergot alkaloid receptor association. Prolonged overstimulation caused by elevated 5-HT is much more difficult to achieve in comparison to ergot alkaloids because of the many mechanisms that control 5-HT neuron firing [7]. However, there is no evidence to show if a controlled increase in 5-HT can compete with ergot alkaloids for receptors to offset and reduce the overstimulation caused by ergot alkaloids. The potential uses of 5-HT as agonist therapy for ergotism in cattle have not been evaluated previously. Therefore, the objective was to evaluate whether the association of 5-HT and ergovaline in a 24 h pre-incubation could affect the vascular contractile response. tent receptor association with 5-HT in comparison with ergot alkaloid derivatives [6] qualifies 5-HT as a potential molecule for agonist therapy for ergot alkaloid toxicosis. Hypothetically, the increase of 5-HT in the synaptic cleft could intensify competition with ergot alkaloids at the serotonergic receptors and reduce the overstimulation through a reduction in ergot alkaloid receptor association. Prolonged overstimulation caused by elevated 5-HT is much more difficult to achieve in comparison to ergot alkaloids because of the many mechanisms that control 5-HT neuron firing [7]. However, there is no evidence to show if a controlled increase in 5-HT can compete with ergot alkaloids for receptors to offset and reduce the overstimulation caused by ergot alkaloids. The potential uses of 5- HT as agonist therapy for ergotism in cattle have not been evaluated previously. Therefore, the objective was to evaluate whether the association of 5-HT and ergovaline in a 24 h pre-incubation could affect the vascular contractile response.

#### **2. Results 2. Results**

#### *2.1. Pre-Exposure to Serotonin 2.1. Pre-Exposure to Serotonin*

Bovine lateral saphenous veins were constricted as evidenced by decreased (*p* < 0.05) internal and external diameters after the 24 h pre-incubation with 5-HT at doses of 1 × 10−<sup>7</sup> M or higher (Table 1). However, no difference (*p* > 0.05) was observed in vessel wall thickness. The pre-incubation with 5-HT produced a dose-dependent decrease in vessel contractility when exposed to increasing concentrations of 5-HT (Figure 1). Pre-incubation concentrations of 1 × 10−<sup>6</sup> M 5-HT and higher resulted in contractile responses to 5-HT that were lower (*p* < 0.05) than control (Figure 2A). The previous exposure dose of 1 × 10−<sup>4</sup> and 1 × 10−<sup>5</sup> M 5-HT almost completely suppressed the contractile response of the vessel. The resultant IC<sup>50</sup> determined from a 24 h previous exposure to increasing concentrations of 5-HT was 7.57 × 10−<sup>7</sup> M (Figure 2B). Bovine lateral saphenous veins were constricted as evidenced by decreased (*p* < 0.05) internal and external diameters after the 24 h pre-incubation with 5-HT at doses of 1 × 10−7 M or higher (Table 1). However, no difference (*p* > 0.05) was observed in vessel wall thickness. The pre-incubation with 5-HT produced a dose-dependent decrease in vessel contractility when exposed to increasing concentrations of 5-HT (Figure 1). Pre-incubation concentrations of 1 × 10−6 M 5-HT and higher resulted in contractile responses to 5-HT that were lower (*p* < 0.05) than control (Figure 2A). The previous exposure dose of 1 × 10−4 and 1 × 10−5 M 5-HT almost completely suppressed the contractile response of the vessel. The resultant IC50 determined from a 24 h previous exposure to increasing concentrations of 5-HT was 7.57 × 10−7 M (Figure 2B).

**Figure 1.** Effects of pre-incubation with serotonin (5-HT) at concentrations of 0.1 × 10−8, 1 × 10−7, 1 × 10−6, 1 × 10−5, 1 × 10−4 M on contractile responses in the isolated lateral saphenous vein from cattle exposed to increasing concentrations of 5-HT. Points represent the mean values and vertical bars show the SEM, *n* = 12. **Figure 1.** Effects of pre-incubation with serotonin (5-HT) at concentrations of 0.1 × 10−<sup>8</sup> , 1 × 10−<sup>7</sup> , 1 × 10−<sup>6</sup> , 1 × 10−<sup>5</sup> , 1 × 10−<sup>4</sup> M on contractile responses in the isolated lateral saphenous vein from cattle exposed to increasing concentrations of 5-HT. Points represent the mean values and vertical bars show the SEM, *n* = 12.

**Figure 2.** (**A**) Effect of pre-incubation with serotonin (5-HT) on contractile concentration–response curve represented as area under curve (AUC) relative to control. \* Indicates a difference relative to control (*p* < 0.05). (**B**)The half-maximal inhibitory concentration (IC50) was 7.57 × 10−7 M, *n* = 12. **Figure 2.** (**A**) Effect of pre-incubation with serotonin (5-HT) on contractile concentration–response curve represented as area under curve (AUC) relative to control. \* Indicates a difference relative to control (*p* < 0.05). (**B**)The half-maximal inhibitory concentration (IC50) was 7.57 × 10−<sup>7</sup> M, *n* = 12.

**Table 1.** Dimensions of bovine lateral saphenous veins after a 24 h incubation with buffer containing 5-HT. **Table 1.** Dimensions of bovine lateral saphenous veins after a 24 h incubation with buffer containing 5-HT.


ternal diameter. 3 Wall thickness. \* Indicates a difference relative to control (0 M) by Dunnett test, *p* < 0.05. <sup>1</sup> Internal diameter. <sup>2</sup> External diameter. <sup>3</sup> Wall thickness.

#### *2.2. Pre-Exposure to Ergovaline 2.2. Pre-Exposure to Ergovaline*

Bovine lateral saphenous veins that were pre-incubated with ERV (in a tall fescue seed extract) at doses of 1 × 10−9 M or higher decreased (*p* < 0.05) the internal diameter while doses of 1 × 10−10 M or higher decreased the external diameter of the vessels (Table 2). Similar to 5-HT, the vessel wall thickness was not affected (*p* > 0.05) by a 24 h preincubation with ERV. Like pre-incubation with 5-HT, the pre-incubation with ERV produced a dose-dependent decrease in contractility in vessels incubated with increasing concentrations of ERV (Figure 3). Only the pre-incubation with 1 × 10−11 M ERV produced little effect on vessel contractility, whereas the 1 × 10−7 M pre-incubation dose almost completely suppressed the contraction of the vessel (Figure 4A). The IC50 for ERV from a 24 h exposure was 1.5 × 10−10 M (Figure 4B). Bovine lateral saphenous veins that were pre-incubated with ERV (in a tall fescue seed extract) at doses of 1 × 10−<sup>9</sup> M or higher decreased (*p* < 0.05) the internal diameter while doses of 1 × 10−<sup>10</sup> M or higher decreased the external diameter of the vessels (Table 2). Similar to 5-HT, the vessel wall thickness was not affected (*p* > 0.05) by a 24 h pre-incubation with ERV. Like pre-incubation with 5-HT, the pre-incubation with ERV produced a dosedependent decrease in contractility in vessels incubated with increasing concentrations of ERV (Figure 3). Only the pre-incubation with 1 × 10−<sup>11</sup> M ERV produced little effect on vessel contractility, whereas the 1 × 10−<sup>7</sup> M pre-incubation dose almost completely suppressed the contraction of the vessel (Figure 4A). The IC<sup>50</sup> for ERV from a 24 h exposure was 1.5 × 10−<sup>10</sup> M (Figure 4B).

**Table 2.** Dimensions of bovine lateral saphenous veins after a 24 h incubation with buffer containing ergovaline*.*  **Table 2.** Dimensions of bovine lateral saphenous veins after a 24 h incubation with buffer containing ergovaline.


extract. <sup>2</sup> Internal diameter. <sup>3</sup> External diameter. <sup>4</sup> Wall thickness. \* Indicates a difference relative to control (0 M) by Dunnett test, *p* < 0.05. <sup>1</sup> Tall fescue seed purified extract. 2 Internal diameter. <sup>3</sup> External diameter. <sup>4</sup> Wall thickness.

12.

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**Figure 3.** Effects of pre-incubation with ergovaline (ERV) at concentrations of 0, 1 × 10−11, 1 × 10−10, 1 × 10−9, 1 × 10−8, 1 × 10−7 M on the contractile responses to serotonin (5-HT) in the isolated lateral saphenous vein from cattle. Points represent the mean values and vertical bars show the SEM, *n* = **Figure 3.** Effects of pre-incubation with ergovaline (ERV) at concentrations of 0, 1 × 10−11, 1 × 10−<sup>10</sup> , 1 × 10−<sup>9</sup> , 1 × 10−<sup>8</sup> , 1 × 10−<sup>7</sup> M on the contractile responses to serotonin (5-HT) in the isolated lateral saphenous vein from cattle. Points represent the mean values and vertical bars show the SEM, *n* = 12. × 10−9, 1 × 10−8, 1 × 10−7 M on the contractile responses to serotonin (5-HT) in the isolated lateral saphenous vein from cattle. Points represent the mean values and vertical bars show the SEM, *n* = 12.

**Figure 3.** Effects of pre-incubation with ergovaline (ERV) at concentrations of 0, 1 × 10−11, 1 × 10−10, 1

**Figure 4.** (**A**) Effect of pre-incubation with ergovaline on contractile concentration–response curve represented as area under curve (AUC) relative to control. \* Indicates a difference relative to control **Figure 4.** (**A**) Effect of pre-incubation with ergovaline on contractile concentration–response curve represented as area under curve (AUC) relative to control. \* Indicates a difference relative to control *p* < 0.05. (**B**) The half-maximal inhibitory concentration (IC50) was 1.57 × 10−10 M, *n* = 12. **Figure 4.** (**A**) Effect of pre-incubation with ergovaline on contractile concentration–response curve represented as area under curve (AUC) relative to control. \* Indicates a difference relative to control *p* < 0.05. (**B**) The half-maximal inhibitory concentration (IC50) was 1.57 × 10−<sup>10</sup> M, *n* = 12.

#### *p* < 0.05. (**B**) The half-maximal inhibitory concentration (IC50) was 1.57 × 10−10 M, *n* = 12. *2.3. Pre-Exposure to Combined Serotonin and Ergovaline 2.3. Pre-Exposure to Combined Serotonin and Ergovaline*

*2.3. Pre-Exposure to Combined Serotonin and Ergovaline*  The final assay evaluated the effects of the IC50 concentrations of ERV and 5-HT alone and 5-HT + ERV in combination during a pre-incubation on vessel dimensions (Table 3) and subsequent contractile response to increasing concentrations of 5-HT (Figure 5). There were no interactions (*p* > 0.05) between treatments for blood vessel length, internal diameter, or external diameter. Bovine lateral saphenous veins pre-incubated with 5-HT at the IC50 concentration had greater (*p* < 0.05) length, lower (*p* < 0.05) internal and external diameters, and no difference (*p* > 0.05) in wall thickness. Conversely, those pre-incubated The final assay evaluated the effects of the IC50 concentrations of ERV and 5-HT alone and 5-HT + ERV in combination during a pre-incubation on vessel dimensions (Table 3) and subsequent contractile response to increasing concentrations of 5-HT (Figure 5). There were no interactions (*p* > 0.05) between treatments for blood vessel length, internal diameter, or external diameter. Bovine lateral saphenous veins pre-incubated with 5-HT at the IC50 concentration had greater (*p* < 0.05) length, lower (*p* < 0.05) internal and external diameters, and no difference (*p* > 0.05) in wall thickness. Conversely, those pre-incubated with ERV at the IC50 concentration tended to have a shorter (*p* = 0.081) length with no difference (*p* > 0.05) in internal and external diameter, or wall thickness. The final assay evaluated the effects of the IC<sup>50</sup> concentrations of ERV and 5-HT alone and 5-HT + ERV in combination during a pre-incubation on vessel dimensions (Table 3) and subsequent contractile response to increasing concentrations of 5-HT (Figure 5). There were no interactions (*p* > 0.05) between treatments for blood vessel length, internal diameter, or external diameter. Bovine lateral saphenous veins pre-incubated with 5-HT at the IC<sup>50</sup> concentration had greater (*p* < 0.05) length, lower (*p* < 0.05) internal and external diameters, and no difference (*p* > 0.05) in wall thickness. Conversely, those pre-incubated with ERV at the IC<sup>50</sup> concentration tended to have a shorter (*p* = 0.081) length with no difference (*p* > 0.05) in internal and external diameter, or wall thickness.

with ERV at the IC50 concentration tended to have a shorter (*p* = 0.081) length with no difference (*p* > 0.05) in internal and external diameter, or wall thickness. There was an interaction (*p* < 0.05) between pre-incubation with 5-HT and ERV on the contractile response to increasing 5-HT, as seen in the AUC response. Previous exposure to 5-HT or 5-HT + ERV reduced (*p* < 0.05) the contractile response by an average of 60% while the pre-incubation with ERV at the IC50 concentration decreased the AUC by only 26%. Interestingly, the 24 h pre-incubation with both 5-HT (7.57 × 10−7 M) and ERV (1.57 × 10−10 M) did not potentiate the inhibitory response (Figure 5). There was no interaction (*p* > 0.05) between 5-HT and ERV on the maximum response to norepinephrine (Table 3). The pre-incubation with 5-HT decreased (*p* < 0.05) the blood vessel contractility There was an interaction (*p* < 0.05) between pre-incubation with 5-HT and ERV on the contractile response to increasing 5-HT, as seen in the AUC response. Previous exposure to 5-HT or 5-HT + ERV reduced (*p* < 0.05) the contractile response by an average of 60% while the pre-incubation with ERV at the IC50 concentration decreased the AUC by only 26%. Interestingly, the 24 h pre-incubation with both 5-HT (7.57 × 10−7 M) and ERV (1.57 × 10−10 M) did not potentiate the inhibitory response (Figure 5). There was no interaction (*p* > 0.05) between 5-HT and ERV on the maximum response to norepinephrine (Table 3). The pre-incubation with 5-HT decreased (*p* < 0.05) the blood vessel contractility response to norepinephrine. Conversely, the pre-incubation with ERV did not affect (*p* > 0.05) the response to norepinephrine. There was an interaction (*p* < 0.05) between pre-incubation with 5-HT and ERV on the contractile response to increasing 5-HT, as seen in the AUC response. Previous exposure to 5-HT or 5-HT + ERV reduced (*p* < 0.05) the contractile response by an average of 60% while the pre-incubation with ERV at the IC50 concentration decreased the AUC by only 26%. Interestingly, the 24 h pre-incubation with both 5-HT (7.57 × 10−<sup>7</sup> M) and ERV (1.57 × 10−<sup>10</sup> M) did not potentiate the inhibitory response (Figure 5). There was no interaction (*p* > 0.05) between 5-HT and ERV on the maximum response to norepinephrine (Table 3). The pre-incubation with 5-HT decreased (*p* < 0.05) the blood vessel contractility response to norepinephrine. Conversely, the pre-incubation with ERV did not affect (*p* > 0.05) the response to norepinephrine.

response to norepinephrine. Conversely, the pre-incubation with ERV did not affect (*p* >

0.05) the response to norepinephrine.


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**Table 3.** Area under curve (AUC) of contractile concentration–response curve to increasing concentrations of serotonin (5-HT) and vascular dimensions of bovine lateral saphenous veins pre-incubated with buffer containing 5-HT, ergovaline (ERV), or both 5-HT and ERV. bated with buffer containing 5-HT, ergovaline (ERV), or both 5-HT and ERV. **Treatments <sup>1</sup>** *p***-Value <sup>2</sup> CTRL 5-HT ERV 5-HT +** 

**Table 3.** Area under curve (AUC) of contractile concentration–response curve to increasing concentrations of serotonin (5-HT) and vascular dimensions of bovine lateral saphenous veins pre-incu-

<sup>1</sup> CTRL= only buffer; 5-HT = buffer with serotonin at IC<sup>50</sup> concentration (7.57 × 10−<sup>7</sup> M); ERV = buffer with ergovaline at IC<sup>50</sup> concentration (1.57 × 10−<sup>10</sup> M); 5-HT + ERV = buffer with serotonin (7.57 × 10−<sup>7</sup> M) and ergovaline (1.57 × 10−<sup>10</sup> M). <sup>2</sup> Main effects of serotonin (5-HT), ergovaline (ERV) and interaction (5-HT × ERV). <sup>3</sup> Area relative to control = 100. <sup>4</sup> Contractile response to norepinephrine (NE, 1 × 10−<sup>4</sup> M). <sup>5</sup> Internal diameter. <sup>6</sup> External diameter. <sup>7</sup> Wall thickness. with ergovaline at IC50 concentration (1.57 × 10−10 M); 5-HT + ERV = buffer with serotonin (7.57 × 10−7 M) and ergovaline (1.57 × 10−10 M). 2 Main effects of serotonin (5-HT), ergovaline (ERV) and interaction (5-HT × ERV). 3 Area relative to control = 100. 4 Contractile response to norepinephrine (NE, 1 × 10−4 M). 5 Internal diameter. 6 External diameter. 7 Wall thickness.

**Figure 5.** Effect of pre-incubation with buffer (CTRL), serotonin (5-HT), ergovaline (ERV), and 5-HT + ERV on contractile concentration–response curve to 5-HT in the isolated lateral saphenous vein from cattle, *n* = 13 to 15. **Figure 5.** Effect of pre-incubation with buffer (CTRL), serotonin (5-HT), ergovaline (ERV), and 5-HT + ERV on contractile concentration–response curve to 5-HT in the isolated lateral saphenous vein from cattle, *n* = 13 to 15.

#### **3. Discussion**

**3. Discussion**  This study aimed to perform the first evaluation of the use of 5-HT to mitigate the negative effects of ergot alkaloids on blood vessel contractility following a prolonged ex vivo pre-incubation. The pre-incubation with 5-HT modified the contractility of vessels exposed at physiological concentrations. The results suggest that 5-HT might compete This study aimed to perform the first evaluation of the use of 5-HT to mitigate the negative effects of ergot alkaloids on blood vessel contractility following a prolonged ex vivo pre-incubation. The pre-incubation with 5-HT modified the contractility of vessels exposed at physiological concentrations. The results suggest that 5-HT might compete with ergot alkaloids for serotonergic receptor binding.

with ergot alkaloids for serotonergic receptor binding. The 24 h incubation with 5-HT and ERV represented normal cellular conditions in an animal. It has been established that 5-HT receptors mediate the contraction of the bovine lateral saphenous vein [15,16]. The reduction of vessel diameter occurred at 5-HT doses below physiological concentrations (1 × 10−7 M). Previous studies have found that bovine plasma 5-HT concentrations are close to 1 × 10−6 M [17,18]. Platelets contain a highly efficient transporter that controls 5-HT in the plasma, which represents the free active 5-HT [19]. The prolonged exposure of a static 5-HT concentration during the pre-incubation The 24 h incubation with 5-HT and ERV represented normal cellular conditions in an animal. It has been established that 5-HT receptors mediate the contraction of the bovine lateral saphenous vein [15,16]. The reduction of vessel diameter occurred at 5-HT doses below physiological concentrations (1 × 10−<sup>7</sup> M). Previous studies have found that bovine plasma 5-HT concentrations are close to 1 × 10−<sup>6</sup> M [17,18]. Platelets contain a highly efficient transporter that controls 5-HT in the plasma, which represents the free active 5-HT [19]. The prolonged exposure of a static 5-HT concentration during the pre-incubation may have caused an overstimulation of the receptors causing a persistent contracted state.

may have caused an overstimulation of the receptors causing a persistent contracted state. The reduction of vessel contractility in the myograph is evidence that prolonged overstimulation can decrease the serotonergic receptor response. The concentration of 5- HT near the nerve terminal may substantially alter the activation or desensitization of serotonergic receptors [20]. Chronic infusion of 5-HT produces a residual desensitization of the receptors in many organs [21]. During the contractility evaluation in the myograph, The reduction of vessel contractility in the myograph is evidence that prolonged overstimulation can decrease the serotonergic receptor response. The concentration of 5-HT near the nerve terminal may substantially alter the activation or desensitization of serotonergic receptors [20]. Chronic infusion of 5-HT produces a residual desensitization of the receptors in many organs [21]. During the contractility evaluation in the myograph, the response to high doses of 5-HT applied for 15 min were rapidly washed out. However, the effect of prolonged exposure to 5-HT during the 24 h pre-incubation was not as readily washed out and a drastic reduction of vessel vasoactivity to 5-HT was observed.

Similar to 5-HT, pre-incubation with ERV provoked an intense reduction of the lateral saphenous vein diameter. In contrast, Trotta et al. [5] did not find a difference in the diameter of bovine ruminal or mesenteric blood vessels after a 2 h incubation with 1 × 10−<sup>8</sup> or 1 × 10−<sup>6</sup> M ERV. However, in the current study, the pre-incubation with ERV with lateral saphenous veins decreased the vessel diameter and the contractility at much lower concentrations. The inclusion of a control treatment ensured that the changes in contractility were caused by the ERV and not by loss of viability. The dose-dependent decrease in contractility after pre-incubation with ERV and the occurrence of this effect at a very low dose (1 × 10−<sup>10</sup> M) was evidence that the effect of ergot alkaloids on blood vessels is mediated by both the dose and the time that the receptors are exposed. Without previous exposure, the ergopeptine ergot alkaloids normally reduce the contractility of vessels at concentrations higher than 1 × 10−<sup>7</sup> to 1 × 10−<sup>6</sup> M [4,22,23], which are considerably higher than the ergovaline levels expected in plasma of ruminants consuming ergot alkaloids [24]. The difference in the concentration of ergot alkaloids and the observed responses in the literature indicates that the duration of exposure can influence the ergot alkaloid effect.

Reductions in the contractile response to 5-HT in lateral saphenous veins collected from endophyte-infected tall fescue of grazing steers has been reported [25]. The reduction in the contractile response to 5-HT of vessels can be associated with a reduction of mRNA of both 5-HT receptors [26,27] and calcium channels [26]. The persistent contracted state of the vessels after ergot alkaloid administration and resistance to washout [15] can be caused by the lower receptor dissociation of the ergot alkaloids [6]. However, prolonged exposure to 5-HT also decreased the relaxation of vessels and the contractility in the organ bath, demonstrating that many mechanisms are involved in changing vessel contractility, rather than receptor kinetics alone.

The lower contractility of blood vessels exposed to ergot alkaloids can be partly explained by the reduction of 5-HT receptors [6]. However, the reduction of contractility after chronic exposure to 5-HT, which is easily washed out compared to ergovaline, indicates other mechanisms also act to modify the contractile response. One plausible mechanism is that the overstimulation of serotonergic receptors, either by chronic 5-HT or ergot alkaloid exposure, can alter calcium dynamics and consequently the smooth muscle contractility.

The administration of ergot alkaloids does not affect the maximum contractile response of vessels to KCl [4,5]. The KCl-derived stimulation bypasses G protein-coupled receptors and activates smooth muscle by the activation of voltage-operated Ca2+ channels [28]. Although it is not clear how ergot alkaloids act on smooth muscle to influence the contractile response, it is possible that they act on G protein-coupled receptors and in different parts of the calcium pathway, other than by voltage-operated Ca2+ channels.

Although prolonged 5-HT exposure mimicked the response of ergot alkaloids, the difference in concentration for a similar response was large. The IC<sup>50</sup> concentration for ERV was almost 5000-fold lower than for 5-HT. The chronic infusion of 5-HT produces a residual desensitization of serotonergic receptors in smooth muscle [21]. The prolonged stimulation of 5-HT receptors leads to phosphorylation, which modifies cell surface expression, coupling profiles, and interactions with protein partners, usually leading to blunted signaling [7]. Serotonin has many mechanisms to control synaptic signaling and prevent neural malfunction. Conversely, the ergot derivatives do not have these mechanisms to control receptor stimuli and their long durations of action are not easily attributed to circulating levels [6]. The vasoconstriction caused by ergot alkaloids can provoke several modifications in cattle, including decreased blood flow and increased blood pressure [9,11,29]. These physiologic modifications can result in localized gangrene and the loss of animal productivity [2,10].

The combined incubation with 5-HT and ERV did not potentiate the inhibitory effect on contractility compared with these substances individually. The competition for receptors may have reduced the potential inhibitory activity when they were combined. These results indicate that an increase in 5-HT has the potential to mitigate vasoconstriction caused by ergot alkaloids and the refinement of optimal concentrations is warranted. The

reduction of the contractile response of vascular smooth muscle to norepinephrine after prolonged exposure to 5-HT or ERV indicates that the residual effects of ergot alkaloids can be associated with triggering the G-protein coupled pathways that can be activated by serotonergic as well as adrenergic receptors. By simultaneously administering ERV and a selective agonist for the 5-HT2A receptor, Trotta et al. [5] observed that the increase in the agonist concentration decreased the contraction caused by ERV in ruminal and mesenteric veins. The use of pharmacologically similar agents as a substitute for an undesired drug is considered agonist therapy [30]. Its efficacy has been proven for dependence on many substances, such as nicotine and opioids [31,32]. The kinetics of ergovaline at vascular serotonergic receptors complicates the development of treatment protocols, yet the prophylactic administration of molecules to modify the 5-HT receptors binding might be effective [33].

In contrast with many illicit drugs, ergotism acts directly on monoamine receptors instead of causing an increase of monoamines in the presynaptic terminal [2]. Thus, strategies to avoid receptor binding potentially achieve better results by reducing neuronal overstimulation and prolonged signaling. Ergot alkaloids (e.g., ergotamine) are extensively metabolized by the liver and cleared from the blood by first-pass hepatic metabolism [34,35]. Jaussaud [24] observed a half-life of 23.6 min for ERV in sheep, which suggests that strategies to avoid receptor binding can accelerate its clearance, reducing the neuronal excitation by the circulating ergot alkaloid.

The 5-HT receptors have complex and integrated functions throughout the body. Although many 5-HT receptors can cause vasoconstriction, activation of the 5HT<sup>7</sup> receptor can provoke a hypotensive response [36]. The stimulation of the 5-HT<sup>7</sup> receptor reduces the contraction induced by the 5-HT2A receptor in the isolated abdominal vena cava [36]. It has been shown that the chronic administration of 5-HT can provoke a sustained fall in blood pressure in rats [37]. Therefore, an increase of 5-HT has the potential to activate receptors for relaxation responses, as well as to compete with ergot alkaloids reducing the persistent overstimulation of the receptor with contractile responses.

#### **4. Conclusions**

The increased exposure of blood vessels to 5-HT appears to compete with ergot alkaloids through receptors in the vascular smooth muscle, indicating that the manipulation of 5-HT has the potential to be used as a treatment of the vasoconstrictive effects induced by ergovaline. However, it is not clear how prolonged exposure to 5-HT and ergot alkaloids can reduce receptor sensitivity. This is further complicated by the fact that ergovaline has a 5000-fold higher potency than 5-HT. Future research should focus on the study of mechanisms related to the induction of blood vessel relaxation when under prolonged serotonergic stimulation, test doses of 5-HT or related compounds, and the evaluation of these in animal models.

#### **5. Materials and Methods**

No live animals were involved in the experiments that make up this study; thus, approval from the University of Kentucky Animal Care and Use Committee was not required.

#### *5.1. Tissue Collection*

The cranial branch of the lateral saphenous vein was collected from 14 predominantly Angus heifers (448 ± 21 kg) immediately after slaughter at the University of Kentucky Meats Laboratory using procedures according to Klotz [38]. Veins from nine heifers were used for calculation of IC<sup>50</sup> (12 vessels sections per treatment) and veins from five heifers were used for testing the IC<sup>50</sup> doses (15 vessels sections per treatment). As part of a separate study, the heifers were kept in the barn and maintained on a silage-based diet for 17 days prior to slaughter. Prior to that, they were managed on endophyte-infected tall fescue pastures with the same silage diet for at least 90 days. Segments (10 cm in length) of lateral saphenous vein at the cranial branch were removed and placed in a modified

Krebs–Henseleit buffer for transport (95% O2/5% CO2; pH = 7.4; 11.1 mM D-glucose; 1.2 mM MgSO4; 1.2 mM KH2PO4; 4.7 mM KCl; 118.1 mM NaCl; 3.4 mM CaCl2; 24.9 mM NaHCO3; Sigma Chemical Co., St. Louis, MO), and were kept on ice during transportation from the collection site to the laboratory.

Blood vessels were dissected and the surrounding adipose and connective tissues were removed. The clean vessel segments were sliced into 2 mm cross-sections using an adjustable acrylic tissue matrix (Braintree Scientific, Inc., Braintree, MA, USA). The vessel cross-sections were inspected under magnification (12.5×) for abnormalities (e.g., structural damage incurred during dissection and points of vascular branches), and abnormal sections were discarded and replaced with viable sections.

#### *5.2. Previous Exposure of Vascular 5-HT Receptors*

To simulate the previous exposure of 5-HT receptors in the blood vessels to the treatments, the vein cross-sections were incubated at 37 ◦C in 5% of CO<sup>2</sup> for 24 h in a 12-well culture plate (1 cross section/well) with 5 mL of Krebs–Henseleit buffer containing either control, serotonin, tall fescue seed extract (source of ergovaline; ERV), or a combination of the treatments. The previous exposures were conducted in two separate assays. The first assay determined the IC<sup>50</sup> for 5-HT and the ergovaline-containing extract. The second assay evaluated the effect of IC<sup>50</sup> doses of 5-HT and ergovaline determined in the previous assay, either individually or in combination.

#### *5.3. IC<sup>50</sup> Determination*

To determine the IC50, stock solutions of 5-HT (Sigma-Aldrich, St. Louis, MO) were diluted to the corresponding final working concentrations of 1 × 10−<sup>8</sup> , 1 × 10−<sup>7</sup> , 1 × 10−<sup>6</sup> , 1 × 10−<sup>5</sup> and 1 × 10−<sup>4</sup> M. Working concentration ranges were prepared based on previous bovine vascular bioassay research using 5-HT [3]. Tall fescue seed extract was prepared and purified as described by Ji et al. [39] and Foote et al. [40]. Stock solutions of the extract were based on ERV concentrations (ergovaline + ergovalinine) and were prepared to final working concentrations of 1 × 10−11, 1 × 10−10, 1 × 10−<sup>9</sup> , 1 × 10−<sup>8</sup> and 1 × 10−<sup>7</sup> M. Working concentration ranges were prepared based on previous bovine vascular bioassay studies using ERV [3,41]. The vein segments were incubated with Krebs– Henseleit buffer alone or with working solutions of 5-HT or ERV for 24 h before contractile response evaluation.

#### *5.4. Associated 5-HT and ERV Pre-Exposure*

The blood vessels were incubated for 24 h with: (1) exclusively Krebs–Henseleit buffer; (2) Krebs–Henseleit buffer with 5-HT at IC<sup>50</sup> concentration; (3) Krebs–Henseleit buffer with ERV at IC<sup>50</sup> concentration; (4) Krebs–Henseleit buffer with 5-HT and ERV both at their respective IC<sup>50</sup> concentrations. Following the completion of the 24 h pre-incubation period, the contractile response to increasing concentrations of 5-HT of each vein cross-section was evaluated.

#### *5.5. Vascular Dimensions*

Following the 24 h incubation, cross-sections were removed from treatment-containing buffer and examined using a dissecting microscope (Stemi 2000-C, Carl Zeiss Inc., Oberkochen, Germany) at 12.5× magnification to measure dimensions of the vessels. Vessel cross-sections were then transferred to a treatment-free Krebs–Henseleit buffer until mounting in the myograph chambers.

#### *5.6. Contractile Response*

After the 24 h incubation period of the treatments, blood vessels were mounted onto luminal supports in individual chambers of a multimyograph (DMT 610M, Danish Myo Technology, Atlanta, GA) with 5 mL Krebs–Henseleit buffer and constant gassing (95% O2/5% CO2; pH = 7.4; 37 ◦C). For each myograph experiment, the incubation treatments

were run in duplicate during the determination of 5-HT and ERV IC<sup>50</sup> and run in triplicate during evaluation of the determined IC<sup>50</sup> doses associated with 5-HT and ERV.

The incubation buffer had the same composition of the transport buffer plus 3 × 10–5 M desipramine (Sigma Chemical, Co.) and 1 × 10–6 M propranolol (Sigma Chemical, Co). These compounds were included to inhibit biogenic amine reuptake and non-specific binding to β-adrenergic receptors, respectively. After mounting the blood vessels on the luminal supports in the myograph, an equilibration period was conducted with constant gassing (95% O2/5% CO2; pH = 7.4; 37 ◦C) for 90 min, with buffer changes every 15 min to allow blood vessels to reach a resting tension of approximately 1 g. At completion of the 90 min equilibration period, 1 × 10−<sup>4</sup> M norepinephrine (Sigma Chemical, Co) was added to each chamber and incubated for 15 min. This served as a reference and to confirm vessel responsiveness and viability. Myograph chambers were then emptied and refilled with incubation buffer to remove norepinephrine and allow the vessels to return to an approximate 1 g tension. Once vessels returned to a resting tension, cumulative additions of 5-HT were added to reach the final concentrations of 5 × 10−<sup>8</sup> , 1 × 10−<sup>7</sup> , 5 × 10−<sup>7</sup> , 1 × 10−<sup>6</sup> , 5 × 10−<sup>6</sup> , 1 × 10−<sup>5</sup> , 5 × 10−<sup>5</sup> , and 1 × 10−<sup>4</sup> M to build the contractile response curve. Afterwards, 1 × 10−<sup>4</sup> M norepinephrine was again added to each chamber for evaluation of the final contractile response. Each 5-HT addition occurred in 15 min intervals in order of increasing concentration. Each cycle (addition) was a 9 min treatment incubation period, two 2.5 min buffer washes, a third buffer replacement, and followed by a 1 min recovery period leading into the next 5-HT addition.

#### *5.7. Data Analysis*

Isometric contractions in blood vessels to norepinephrine and 5-HT were digitized and recorded in grams of tension using PowerLab 16/35 and Chart software (version 8, ADInstruments, Colorado Springs, CO, USA). Baseline tensions were recorded before addition of each compound. For all contractile response data, the maximum tension (measured in g) during the 9 min incubation was recorded as the contractile response and corrected for baseline tension.

Due to variation between animals and tissues, contractile response data were normalized as a percentage of the maximum of tension induced by the reference addition of norepinephrine to compensate for differences in vessel responsiveness. Vessel contractile response data were reported as the percentage mean contractile response ± SEM of the maximum contractile response produced by the 1 × 10−<sup>4</sup> M norepinephrine reference addition in vessels incubated for 24 h without treatment compounds. All contractile response data presented were plotted using GraphPad Prism software (San Diego, CA, USA). Sigmoidal concentration response curves to each treatment were calculated and plotted using a nonlinear regression equation:

$$\mathbf{y} = \frac{100}{1 + 10^{\ (\mathbf{b} - \mathbf{X}) \ast \mathbf{a}}}$$

where y is the contractile response, X is the concentration of the test solution, a is the Hill slope, and b is the concentration corresponding to the response midway between bottom and top (IC50).

#### *5.8. Statistical Analysis*

All data were analyzed as a completely randomized design using the MIXED model of SAS with the fixed effect of previous exposure treatment and the random effect of the incubation run.

The area under curve (AUC) was calculated using a linear trapezoidal method with baseline set to zero. The AUC was transformed, setting the control value to 100. Comparison of AUC and dimension of vessels to control in previous exposure doses was conducted by Dunnett test.

The heifer from which the vessel was collected and the myograph run were included as random effects to compare associated 5-HT and ERV previous exposure. Pairwise comparisons were conducted using the LSD test. Statistical significance was considered at *p* ≤ 0.05 and tendency at 0.05 < *p* ≤ 0.10.

**Author Contributions:** Conceptualization, E.E.L.V., D.L.H., J.L.K.; formal analysis, E.E.L.V., D.L.H., J.L.K.; investigation, E.E.L.V., J.L.K.; writing—original draft preparation, E.E.L.V., D.L.H., J.L.K.; writing—review and editing, E.E.L.V., D.L.H., J.L.K.; funding acquisition, J.L.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work is funded by Hatch Capacity Grant Project no. KY007088 from the USDA National Institute of Food and Agriculture and Project 201807121511 from USDA/ARS and the University of Kentucky Agricultural Experiment Station.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors acknowledge Adam J. Barnes of the Forage-Animal Production Research Unit (Lexington, KY, USA) for his technical assistance.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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