The present investigation was designed to investigate the use of blessed thistle (Cnicus benedictus) in amelioration of fescue toxicosis in beef cattle. Twenty-seven crossbred Angus cows were blocked by age and weight into groups of three. Each group (n=9) was then randomly assigned to one of three dietary treatments: endophyte-free (EF), endophyte-infected (EI), or endophyte-infected with addition of cut and stemmed blessed thistle (EIBT). Each group was also randomly assigned to one of three 29-day replicates. Parameters measured during this trial included: urinary ergot alkaloid concentration (ng ergot alkaloids/mg creatinine), body weight (kg), rectal temperature (°C), serum progesterone (ng/ml), and serum prolactin (ng/ml). No effect of treatment (P>0.05) was detected in any of these parameters. A treatment x time interaction (P<0.0001) was observed for progesterone concentrations, likely a result of estrous cyclicity of cows. Results of this study suggest that we were unsuccessful in inducing fescue toxicity in test subjects assigned to EI and EIBT treatments. Minimal dietary ergovaline (1.72 μg kg-1) and cool ambient temperatures (-6.7°C to 23.3°C) likely contributed to this inability. Data from this research do suggest, however, that C. benedictus can be successfully incorporated into cattle diets. Further research will need to be performed to fully elucidate what, if any, benefits blessed thistle can have in the treatment of fescue toxicity.

Fescue toxicosis is problematic for ruminant livestock, causing weight loss and low productivity when fed endophyte-infected forages. Complete underlying mechanisms of toxicosis are unknown therefore; the objective of the study was to determine if ruminally dosed ergot alkaloids impact rumen motility. Cannulated steers were pair-fed a forage diet and ruminally dosed with endophyte-free (E-) or endophyte-infected (E+) tall fescue seed. An 8-h period of rumen motility collection began 4-h after feeding by monitoring pressure change via a wireless telemetry and transducer system. In experiment 1, steers were paired by weight and assigned to E- or E+ treatment. Overall, E+ steers had more frequent contractions. On d 7 - 9, both treatments had lower frequencies and E- steers had greater amplitude of contractions, which corresponded with decreased DM intake. In experiment 2 steers remained in pair, but switched treatment. During the 57 d E+ steers received titrated levels of ergovaline + ergovalinine. There was no difference between treatments for frequency or amplitude of contractions, but increasing dosage, decreased frequency (d 1 - 44) and amplitude, coinciding with lower DM intakes. Alteration in rumen motility associated with changes in intake may be responsible for the decreased productivity in ruminants consuming E+ forages.

Tall fescue is a widely grown, cool season grass prevalent in the eastern United States that is known for its resistance to abiotic and biotic stresses. A main reason for tall fescue's resistance to these stresses is attributed to the presence of a fungal endophyte. Unfortunately, this endophyte also adversely affects cattle production. Cows consuming the ergot alkaloids produced by these endophytes can exhibit decreased feed intake, growth performance, organ vasoconstriction, and increased rectal temperature. This work is interested in examining how endophyte toxin exposure impacts pregnancy in cattle. Reduced blood flow to the fetus and inadequate maternal nutrition contributes to intra uterine growth restriction (IUGR), and this work proposed that fescue endophyte toxicity affects the gestating cow and fetus. Three studies were completed. In experiment 1, gestating cows grazed high or low endophyte fescue pastures during late gestation to determine if exposure to ergot alkaloids in utero results in IUGR and if calves from these pregnancies have altered growth performance. Creep feeding was evaluated as a mitigation strategy for impaired calf growth due to fescue toxicity, and feedlot performance was evaluated to determine if consuming fescue during gestation and creep feeding would affect feedlot performance. Calf BW was different (P < 0.01) by treatment x time. Birth weights of calves were similar , prior to creep feeding calves exposed to high endophyte fescue were lower, and post-supplementation creep fed calves had increased BW. Days on feed and dressing percentage were decreased in the supplemented group, and marbling score was decreased for both the supplemented and unsupplemented groups following the completion of the feedlot phase (P < 0.05). The second study was setup similar to study one, however cows were exposed to fescue pastures from d 170 of gestation until calving. Calf birth weights did not differ, but weights were increased in the supplemented group post creep feeding (P < 0.05). Average daily gains (ADG) of supplemented calves were greater during the supplementation period (P < 0.01). In the third study, indwelling vaginal temperature probes were used to evaluate differences in body temperature of cows fed fescue seed with high or low levels of ergot alkaloids during early gestation, and in varying environmental conditions. In the winter trial, body temperature was measured hourly from days 0-14 of gestation. In the summer trial, body temperature was measured hourly from days 0-32 of gestation. Body temperatures were different (P < 0.01) between treatments during both trials. / Master of Science

Silvopastures integrate trees, forages, and livestock. Tall fescue, the dominant forage in much of the U.S., harbors an endophyte that produces toxic ergot alkaloids. Diluting the sward with other forages can reduce ergot alkaloid concentrations, but it is unknown how shade impacts alkaloid production and productivity of different forage mixtures. This study tested the effects of increasing shade and mixture complexity on sward yield, botanical composition, nutritive characteristics and ergot alkaloid concentrations. Slatted shade structures created 30, 50, and 70% shade compared to full sun. Three forage mixtures were evaluated (SIMPLE = tall fescue and white clover; INTERMEDIATE = SIMPLE + orchardgrass and red clover; and COMPLEX = INTERMEDIATE + Kentucky bluegrass, birdsfoot trefoil, and alfalfa). Fifty and 70% shade reduced yield while red clover and orchardgrass dominated shaded swards. Birdsfoot trefoil, Kentucky bluegrass, and white clover did not perform well in any treatment. Nutritive value declined beneath shade in spring and fall. Sward ergot alkaloid concentration increased beneath shade in simple mixtures because of greater proportions of tall fescue. In the intermediate and complex mixtures, ergot alkaloids were diluted by other forage species and was not affected by shade. This illustrates the importance of incorporating multiple species into the sward. Low light levels may not have been sufficient to meet the forages' high energetic demands in the spring. Even though total forage production or nutritive value may be sacrificed during part of the year, this may be compensated for by diverse swards diluting ergot alkaloid concentrations. / Master of Science

Two experiments were conducted at the University of Kentucky, Eden Shale Farm, Owenton, KY to evaluate the use of MTB-100TM (Alltech, Inc., Nicholasville, KY) to alleviate the symptoms of fescue toxicity when lactating Angus x Beefmaster cows and their calves grazed endophyte-infected KY-31 tall fescue. Experiment 1 provided a carbohydrate based toxin adsorbent, MTB-100TM, ad libitum in a commercial mineral supplement to project a daily consumption rate of 0, 20 or 40 g of MTB-100TM per cow. Cows were weighed, assigned a body condition score (BCS) and hair coat score (HC), rectal temperatures were recorded and fecal grab samples were taken for ergovaline (EV) and lysergic acid (LA) analysis every 35 days for three grazing seasons (May to September). Calves were also weighed and assigned a HC score. Although MTB-100TM did not improve cow or calf performance, cows older than 4 years and those with greater Beefmaster breeding exhibited a higher tolerance to fescue toxicity than 2 and 3-yr-olds and cows with greater Angus breeding. Experiment 2 was conducted to evaluate the response of lactating beef cows and their calves to strategic supplementation with MTB-100TM. MTB-100TM was mixed with a complete mineral so daily intake was projected to be 0 or 20 g/cow. The experimental period extended from May 5 to October 2 and was divided into 3 strategic periods: P1 = May 5 to July 5; P2 = July 5 to August 31; P3 = August 31 to October 2. Treatments were either 0 or 20 g•cow-1•d-1 MTB-100TM within a period (Treatment 1 = 0, 0, 0; Treatment 2 = 20, 0, 20; Treatment 3 = 0, 20, 0; Treatment 4 = 20, 20, 0; and Treatment 5 = 20, 20, 20). Cow and calf performance was measured the same as Exp. 1, but every 21 days. Supplementation early in the grazing season tended to improve cow weight gain and body condition; however, there was no effect on calf performance. Fecal output of EV and LA did not increase in either experiment with supplementation. In conclusion, strategically invoked MTB-100TM consumption can increase performance of cows grazing endophyte-infected tall fescue forage.

The first goal of the current research was to determine whether gene expression profiles differed between whole pituitaries of growing beef steers grazing pastures containing high (HE) or low (LE) amounts of toxic endophyte-infected tall fescue. The global (microarray analysis) and selected targeted (RT-PCR) mRNA expression patterns of pituitaries collected from beef steers (BW = 266 ± 15.5 kg) that had been randomly assigned to undergo summer-long grazing (89 to 105 d) of either HE (0.52 ppm ergot alkaloids) or LE (< 0.03 ppm ergot alkaloids) pastures were compared. Gene expression data were subjected to one-way ANOVA. The pituitaries of HE steers had 542 differentially expressed genes, and the pattern of altered gene expression was dependent on treatment. Targeted RT-PCR analysis corroborated these findings, including decreased expression of DRD2, PRL, POU1F1, GAL, and VIP and that of POMC and PCSK1, respectively. Canonical pathway analysis (Integrated Pathway Analysis, IPA) identified HE-dependent alteration in signaling of additional pituitary-derived hormones, including growth hormone and GnRH. In conclusion, consumption of endophyte-infected tall fescue alters the pituitary transcriptome profiles of steers in a manner consistent with their negatively affected physiological parameters. The second goal of this project was to test the hypothesis that sodium selenite (ISe), SEL-PLEX (OSe), vs. a 1:1 blend (MIX) of ISe and OSe in a basal vitamin-mineral (VM) mix would differentially alter pituitary transcriptome profiles in growing beef steers (BW = 183 ± 34 kg) commonly grazing an endophyte-infected tall fescue (HE) pasture. Steers were randomly selected from herds of fall-calving cows grazing HE pasture and consuming VM mixes that contained 35 ppm Se as either ISe, OSe, or MIX forms. Steers were weaned, depleted of Se for 98 d, and subjected to summer-long common grazing of a 10.1 ha HE pasture containing 0.51 ppm ergot alkaloids. Steers were assigned (n = 8) to the same Se-form treatments on which they were raised. Selenium treatments were administered by daily top-dressing 85 g of VM mix onto 0.23 kg soyhulls, using in-pasture Calan gates. Pituitaries were collected at slaughter and changes in global (microarray) and selected (RT-PCR) mRNA expression patterns determined. The effects of Se treatment on relative gene expression were subjected to one-way ANOVA. The form of Se affected the expression of 542 annotated genes. Integrated Pathway Analysis found a canonical pathway network between prolactin and POMC/ACTH/ α-MSH synthesis-related proteins, and that mitochondrial dysfunction was a top-affected canonical pathway. Targeted RT-PCR analysis found that the relative abundance of mRNA encoding prolactin and POMC/ACTH/ α-MSH synthesis-related proteins was affected by the form of Se, as were mitochondrial dysfunction-related proteins OSe steers appeared to have a greater prolactin synthesis capacity vs. ISe steers through decreased dopamine receptor D2 signaling, whereas MIX steers had a greater prolactin synthesis capacity and release potential by increasing TRH concentrations than ISe steers. OSe steers also had a greater ACTH and α-MSH synthesis potential than ISe steers. We conclude that form of Se in VM mixes affected genes responsible for prolactin and POMC/ACTH/α-MSH synthesis, and mitochondrial function in pituitaries of growing beef steers commonly grazing an HE pasture. The third goal was to test the hypothesis that sodium selenite (ISe), SEL-PLEX (OSe), vs. a 1:1 blend (MIX) of ISe and OSe in a basal vitamin-mineral (VM) mix would differentially alter selenoprotein profiles in pituitaries and livers of growing beef steers commonly grazing an endophyte-infected tall fescue (HE) pasture (i.e., the same steers used in Goal 2). The effects of Se treatment on relative gene expression were subjected to one-way ANOVA. The mRNA content of 6 selenoproteins in the pituitary was affected by Se treatments, along with two selenoprotein P receptors, whereas the expression of two selenoproteins was altered in the liver. We conclude that the change in selenoprotein gene expression in pituitaries indicates that OSe steers have a greater potential capacity to manage against oxidative damage, maintain cellular redox balance, and have a better quality control of protein-folding in their pituitaries than ISe steers. The change in selenoprotein gene expression by the liver indicates that MIX steers have a greater redox signaling capacity and capacity to manage oxidative damage than ISe steers.

Consumption of endophyte-infected (E+) grasses has long been associated with health problems in animals. In cattle E+ tall fescue consumption leads to fescue toxicosis, and in horses it leads reproductive problems. The health-related issues associated with endophyte consumption have been attributed to the effects caused by the ergot alkaloids produced by the fungus. These ergot alkaloids are considered D2-like receptor agonists, and 5-HT2 serotonin and α-adrenergic receptor partial agonists. Many studies in humans, swine, cattle, and horses have identified that ergopeptines cause a decrease in prolactin production due to their dopaminergic activities. Additionally, these molecules have been found to cause vasoconstriction in cattle and horses through their other agonistic activities. Furthermore, dopamine agonists are currently being used to treat pituitary pars intermedia dysfunction (PPID) in horses, a condition in which the horse lacks sufficient dopamine. However, the ergot alkaloids found in E+ tall fescue had not previously been investigated for their potential benefits in treating PPID horses. Moreover, little research has investigated the effects of ergot alkaloids and dopamine agonists on the immune system of horses, even though many health problems associated with E+ tall fescue consumption suggest there to be an elicited inflammatory response. Thus, the primary objective of this study was to establish an understanding of immune and hormone responses to ergot alkaloids and dopamine agonists in the horse. The hypothesis of this body of research was that ergot alkaloids and bromocriptine both would elicit inflammatory and hormone responses in the horse. Specifically, this research was conducted to determine the effects of E+ tall fescue seed consumption on immune, hormone, and vasoconstrictive responses, in both non-PPID and PPID horses. In addition, both the in vitro and in vivo effects of bromocriptine on cytokine production from equine peripheral blood mononuclear cells (PBMCs) were investigated. In the first study, there were no significant changes in body morphometrics, vasoconstriction, hormone responses or cytokine expression due to the consumption of ergot alkaloids in non-PPID and PPID horses. The second study was an in vitrostudy in which PBMCs were exposed to varying concentrations of either bromocriptine, a D2-like receptor agonist that is used as a model for ergot alkaloid consumption, or dopamine. This experiment demonstrated that exposure to dopamine or a dopamine agonist at a concentration greater than 10-5M is toxic to PBMCs, and that bromocriptine elicits an anti-inflammatory effect at concentrations less than 10-5M. Concentrations of dopamine less than 10-5M, on the other hand, did not cause any significant changes in cytokine expression. A third study was conducted that evaluated the effects of an intravenous injection of bromocriptine on hormone and immune responses in the aged mare. This study identified that bromocriptine maximally reduced prolactin levels 12 hours post-injection and prolactin returned to baseline levels approximately 56 hours post-injection. Additionally, only a significant increase in IL-1β was detected 12 hours post-injection, which suggests bromocriptine was activating an innate immune response. Overall, the body weights and rectal temperatures of horses did not significantly change in any of the experiments, which indicated that aged non-pregnant horses are able to tolerate E+ tall fescue. In addition, this body of research identified that intravenous delivery of a semi-synthetic dopamine agonist, bromocriptine, and not an oral delivery of an E+ tall fescue seed derived dopamine agonist, caused a decrease in prolactin concentrations, but revealed conflicting results regarding inflammatory responses. In summary, further research is warranted to determine the mechanism of action that dopamine agonists have on the immune system of horses.

Graduation date: 1998

Endophytic fungi (A. coenophialum and A. lolii) which infect grasses produce ergot alkaloids that serve as the grasses' chemical defenses and enhance the vigor of the grass. Turf-type tall fescue with high endophyte levels has been deliberately developed to produce a greener, more vigorous, pest-resistant turf. Consumption of endophyte-infected grass causes various toxicity symptoms in livestock. Cattle in the southeastern and midwestern United States, where tall fescue is grown on 14 million hectares, often develop signs of toxicosis during summer months from grazing plants in fected by A. coenophialum. A more severe form of the disease, fescue foot, has been associated with cold environment and reported in late fall and winter months not only in the southeastern United States but also in the northwest United States. In New Zealand, where perennial ryegrass is grown on 7 million hectares of pasture, sheep often develop a condition called ryegrass staggers from grazing plants infected by A. lolii. New Zealand reports economic losses grazing plants infected by A. lolii. New Zealand reports economic losses associated with the sheep industry of $205 million per year. In the United States, economic losses associated with the beef cattle industry alone is estimated at $600 million per year. Range finding experiments and case studies of fescue foot and perennial ryegrass staggers (PRGS) were conducted on cattle and sheep under grazing and barn conditions. The main objective was to determine threshold levels of the endophyte toxins, ergovaline (EV) (appendix 1) and lolitrem B (appendix 2), associated with the diseases of fescue foot and PRGS respectively. Fescue foot was experimentally induced in cattle under barn studies in the spring with 825 ppb ergovaline. The ergovaline contaminated feed was given for a period of 42 days. Similar barn studies in sheep in spring to early summer did not produce clinical fescue foot with up to 1215 ppb. Field studies of natural fescue foot in a herd of sheep were conducted, (ie 540 ppb) values of ergovaline in the feed, but clinical disease was not produced in late fall through winter. A case study from a herd of sheep revealed 813 ppb dietary ergovaline had produced fescue foot in the months of fall (November). Fields of perennial ryegrass (PRG) where sheep received 2,135 ppb lolitrem B toxin were associated with clinical cases of PRGS in 42 sheep of 237 sheep (18 percent incidence rate) in the Willamette Valley of Oregon. Three months later, sheep on this same field which then had 1,465 ppb lolitrem B, did not have PRGS. These were the first range finding experiments undertaken in this locale to document threshold levels of endophyte toxins associated with fescue foot and PRGS. / Graduation date: 1994

Twenty-four steers were grazed on endophyte (Neotyphodium coenophialum)-infected [[Lolium arundinaceum (Schreb.) Darbysh]; E+] tall fescue (TF) and exposed to ergot alkaloids for an 106-d grazing period. Cattle were removed from pasture, placed in dry lots, and fed a non-toxic diet to evaluate changes in vascular contraction relative to time-off E+ TF pasture. Lateral saphenous veins (SV) were biopsied from steers at 0-, 21-, 42-, and 63-d off TF pasture and from 6 control steers at 0- and 63-d off bermudagrass (BG) pasture. To evaluate contractile response, biopsied SV were exposed to increasing concentrations of ergotamine in a multimyograph. Cross-sectional scans of the caudal artery were taken using color Doppler ultrasonography on 0-, 8-, 15-, 21-, 29-, 36-, 42-, and 45-d to determine artery luminal area. Contractility of SV was less for TF than BG steers on d 0, but was similar between the two treatments by d 63. Luminal areas of the caudal arteries in E+ TF steers relaxed over time and were similar to BG steers by 36 d off pastures. Results indicated that alkaloid-induced vasoconstriction in cattle grazed on E+ TF can be relaxed in 5 to 6 weeks after they are placed on non-toxic diets.