transport across ruminant gastric tissues N. S. Hill, F. N. Thompson, J. A. Stuedemann, G. W. Rottinghaus, H. J. Ju, D. L. Dawe and E. E. Hiatt, 3rd

J Anim Sci 2001. 79:542-549.

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Downloaded from jas.fass.org at USDA Natl Agricultural Library on December 17, 2008. Ergot alkaloid transport across ruminant gastric tissues

N. S. Hill*,1, F. N. Thompson†, J. A. Stuedemann‡, G. W. Rottinghaus§, H. J. Ju*, D. L. Dawe†, and E. E. Hiatt, III*

*Department of Crop and Soil Sciences, †College of Veterinary Medicine, University of Georgia, Athens 30602; ‡USDA-ARS J. Phil Campbell Sr. Natural Resource Center, Watkinsville, GA 30677; and §College of Veterinary Medicine, University of Missouri, Columbia 65211

ABSTRACT: Ergot cause fescue toxicosis measured and the potential transportable alkaloids cal- when livestock graze endophyte-infected tall fescue. It culated by multiplying the moles of transported alka- is generally accepted that is the toxic compo- loids per square centimeter of each tissue type by the nent of endophyte-infected tall fescue, but there is no surface area of the tissue. Studies were conducted to direct evidence to support this hypothesis. The objective compare alkaloid transport in reticular, ruminal, and of this study was to examine relative and potential omasal tissues and to determine whether transport was transport of and ergopeptine alkaloids across active or passive. Ruminal tissue had greater ergot al- isolated gastric tissues in vitro. Sheep ruminal and kaloid transport potential than omasal tissue (85 vs 60 omasal tissues were surgically removed and placed in mmol) because of a larger surface area. The ruminal parabiotic chambers. Equimolar concentrations of ly- posterior dorsal sac had the greatest potential for alka- sergic acid, lysergol, ergonovine, , and ergo- loid transport, but the other ruminal tissues were not cryptine were added to a Kreb’s Ringer phosphate (KRP) solution on the mucosal side of the tissue. Tissue different from one another. Alkaloid transport was less was incubated in near-physiological conditions for 240 among reticular tissues than among ruminal tissues. min. Samples were taken from KRP on the serosal side Transport of alkaloids seemed to be an active process. of the chambers at times 0, 30, 60, 120, 180, and 240 The alkaloids with greatest transport potential were min and analyzed for ergot alkaloids by competitive and lysergol. Ergopeptine alkaloids tended ELISA. The serosal KRP remaining after incubation to pass across omasal tissues in greater quantities than was freeze-dried and the alkaloid species quantified by across ruminal tissues, but their transport was minimal HPLC. The area of ruminal and omasal tissues was compared to lysergic acid and lysergol.

Key Words: Ergot Alkaloids, Festuca, Rumen Mucosa, Toxicity

2001 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2001. 79:542–549

Introduction (1998) found that approximately 94% of the alkaloids from cattle grazing endophyte-infected tall fescue al- Livestock grazing endophyte-infected tall fescue in- kaloids were found in the urine and 6% in the bile. gest ergot alkaloids that cause the condition known Extensive research using nonruminants suggests that as fescue toxicosis (Hill et al., 1994; Bouton et al., ergopeptine alkaloids are excreted via the bile and 1998). Ergot alkaloids are grouped into two broad ergoline alkaloids are excreted through the urine (see classes: the ergoline alkaloids that contain the lysergic reviews by Nimmerfall and Rosenthaler, 1976; Eckert ring structure with hydroxyl, carboxyl, or carboxamide et al., 1978; Griffith et al., 1978). Assuming that the functional groups and the ergopeptine alkaloids that metabolic functions of the excretory organs are highly have a tripeptide cyclol moiety attached at the carbox- conserved among ruminants and nonruminants, these amide site (Rutschmann and Stadler, 1978). studies suggest that the circulating alkaloids in rumi- Little is known about bioavailability or metabolism nants grazing tall fescue are either 1) converted from of the ergot alkaloids in ruminants. Stuedemann et al. the ergopeptine form before excretion, 2) absorbed as the ergoline alkaloids, or 3) absorbed as both and me- tabolized to the ergoline prior to excretion. Urinary appearance and disappearance of ergot al- 1 Correspondence: 3111 Miller Plant Sciences Bldg. (phone: 706- kaloids occurs within 12 h following switching animals 542-0923; fax: 706-542-0914; E-mail: [email protected]). Received March 24, 2000. among endophyte-infected and endophyte-free pas- Accepted October 27, 2000. tures (Stuedemann et al., 1998). Expeditious appear-

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Downloaded from jas.fass.org at USDA Natl Agricultural Library on December 17, 2008. Ergot alkaloid transport across tissues 543 ance and disappearance of urinary alkaloids is most Ergot alkaloids used in the experiments were a mix- likely to occur if the alkaloids are soluble in ruminal ture of lysergol, lysergic acid, ergonovine, ergocryp- fluids. Because the liquid fraction of the digesta is tine, and ergotamine tartrate. Lysergol, lysergic acid, associated with the ruminal microbial ecosystem or is and ergonovine were selected because they represent recycled in the omasum (Church, 1979; Ruckebusche the , acid, and amide forms of the ergoline alka- and Thivend, 1980), these tissues may serve as an loids, respectively. and ergotamine tar- important absorptive site for the alkaloids. The objec- trate were used as the ergopeptine alkaloids. Each tive of this study was to ascertain which forms of ergot alkaloid had a final concentration of 30.5 mM in etha- alkaloids are absorbed from the digestive system by nol. Equimolar concentrations of each alkaloid were ruminal and omasal tissues. used so that transport affinities of each could be as- sessed. One-tenth milliliter of alkaloid solution was Materials and Methods administered to the mucosal chamber. Thus, the final concentration of each alkaloid in the mucosal cham- All experiments were conducted using tissues from bers was 30.5 ␮M in 10 mL of solution of the mucosal digestive tracts of Rambouillet ewe lambs (Ovis aries). side of the chamber. The serosal chamber also received The ewes were approximately 8 mo old and were main- 0.1 mL of ethanol to maintain equal osmotic potential. tained at the USDA-ARS J. Phil Campbell Sr. Natural Mucosal chambers not receiving ergot alkaloids re- Resource Center, Watkinsville, GA in compliance with ceived 0.1 mL of ethanol to maintain uniform test con- animal health and well-being guidelines set by USDA ditions. and the University of Georgia. Ewes grazed bermu- Experiment 1: Ergot Alkaloid Transport in Ruminal dagrass pasture and shade, water, and salt blocks con- and Omasal Tissues. Tissues from two ewes were used taining trace minerals were available at all times. The for this study: each ewe served as a tissue donor on a ewes were killed using a captive bolt before tissues separate day. Four parabiotic chambers were prepared were excised for alkaloid transport analyses. using ruminal posterior ventral sac and tissue from In vitro transport of ergot alkaloids from mucosal omasal plies from each animal. Two chambers for each to serosal sides of the tissues was performed using tissue type were randomly assigned to one of two water parabiotic chambers as described by Matthews and baths. Tissue types within each water bath were ran- Webb (1995). Ruminal, reticular, and omasal compart- domly assigned to alkaloid 180 and 240 min after ad- ments were surgically removed from animals within ministration of the alkaloids to the mucosal chamber 5 min of death. Ruminal, reticular, and omasal com- by aspirating 300 ␮L of solution. Alkaloid appearance partments were emptied of their contents by inverting in the serosal chamber was determined using a com- the chambers, rinsing them clean with tap water, and petitive ELISA procedure as described by Adcock et transporting them to the laboratory in 0.85% saline al. (1997). β-Hydroxybutyric acid was determined en- solution. Tissues were received in the laboratory and zymatically in serosal solutions (Gau, 1987) to verify immediately placed in Kreb’s ringer phosphate (KRP) tissue viability. After the 240-min sample was taken, solution (Umbreit et al., 1964). Ruminal and reticular a 1.5-mL volume of mucosal and serosal buffer was tissues were prepared by removing the musculature aspirated from the chambers and lyophilized. Concen- from the serosal sides of the tissues and placing them trations of ergot alkaloids were determined in the ly- in KRP with 10 mM glucose (KRPG) prior to place- ophilized serosal buffer using HPLC (Rottinghaus et ment in parabiotic chambers. Omasal tissue was pre- al., 1993) to determine tissue transport. pared by peeling plies in half to expose the serosal The remaining tissues from the rumen, reticulum, sides of the tissue before placing it in the KRPG. and omasum were cut into sections and traced on 21.6- A 95:5 mixture of oxygen:carbon dioxide was bub- × 27.9-cm plain white paper. The outlines of the tissue bled through KRPG buffer for 60 min in a water bath were cut and the paper pieces representing the area maintained at 39°C prior to each study. Tissues sec- of each tissue type were dried at 70°C for 16 h in a tions, approximately 2 × 2 cm, were placed into the convection oven and weighed. A 120-cm2 section of parabiotic chambers, rubber O-rings were placed on paper was cut, dried, and weighed to obtain a standard the mucosal side of the tissues, and the chambers were weight per unit area for the paper. The paper pieces sealed with thumb-screw clamps. Each side of the representing the tissues were weighed, and the area chamber was filled with 10 mL of the gassed KRPG of the paper was calculated to estimate the relative buffer and placed in a water bath maintained at 39°C. area (not accounting for papillae or villi) of the tis- Butyric acid was added to the mucosal side of the sue surfaces. chamber at a concentration of 15 mM. A 95:5 mixture A factorial arrangement of tissue type (ruminal or of oxygen:carbon dioxide gas was bubbled through the omasal) by ergot alkaloid treatments (no alkaloids vs solutions on both sides of the chamber for the duration added alkaloids) was assigned randomly to a complete of the experiments. Approximately 30 min lapsed from block design; two water baths served as replications. the time of killing of animals to placement of parabiotic Therefore, data from two replications were gathered chambers containing prepared tissues into the water on d 1 using tissues from one ewe and data from two bath. replications were gathered on d 2 using tissues from

Downloaded from jas.fass.org at USDA Natl Agricultural Library on December 17, 2008. 544 Hill et al. a second ewe. Analysis of variance was conducted us- Table 1. Relative surface areas and potential ing a factorial arrangement of tissue type and ergot alkaloid transport of reticular, ruminal, alkaloid treatments with a split plot in time (Snedecor and omasal tissues in sheep and Cochran, 1978) analyzed by PROC GLM of SAS 2 (SAS Inst. Inc., Cary, NC). Treatment means were Tissue type Relative area, cm % of Total area separated using Fisher’s protected LSD. Following Reticulum 316b 8.85c verification of differences for alkaloid transport using Rumen analysis of variance, mean ELISA values for the sero- Anterior dorsal 453a 12.69ab sal data for each tissue type (dependent variable) were Posterior dorsal 543a 15.21a a ab regressed against time (independent variable) using Anterior ventral 461 12.91 Posterior ventral 434a 12.16b linear and quadratic models with the PROC REG sub- RMSE 46 0.93 routine of SAS (SAS Inst. Inc.). Total rumen 1,891a 52.98a Experiment 2: Ergot Alkaloid Transport Across Rumi- Omasum 1,362b 38.17b nal and Reticular Tissues. Tissues from two ewes were RMSE 32 1.53 used for this study, and each ewe served as a tissue a,b,cMeans within a column within a grouping with different super- donor on a separate day. Posterior and anterior sec- scripts differ (P < 0.05). tions of the ventral and dorsal sacs of the rumen and the dorsal and ventral sections of the reticulum were used. As with the first experiment, two parabiotic determined in serosal samples via enzymatic assay chambers were prepared for each tissue type daily, with one chamber randomly assigned to one of two (Gau, 1987) to verify whether the tissues were living water baths. Administration of alkaloids, sampling of or dead. the parabiotic chambers, and analysis of alkaloids A factorial of tissue type (ruminal or omasal) by were conducted as in Exp 1. Potential alkaloid trans- sodium azide treatment (no azide vs 0.2% azide) was port was calculated using values for the areas of each assigned within each of two waterbaths. The wa- of the tissues obtained in Exp 1. terbaths served as blocks; two replications of each Analysis of variance was conducted using a random treatment were conducted from tissues excised from assignment of tissue type within each block with re- each ewe. Therefore, data from two replications were peated measures of alkaloid transport made in time gathered on d 1 and data from two replications were (Snedecor and Cochran, 1978). Analysis of variance gathered on d 2. Analysis of variance was conducted was conducted to determine whether differences oc- using a factorial arrangement of tissue type and so- curred among tissue types, time, or tissue types × time dium azide treatments within each block and repeated interaction. Treatment variables were separated using measures of alkaloid transport made in time (Snedecor Fisher’s protected LSD. Regression analysis was con- and Cochran, 1978). Treatment variables with signifi- ducted on means of ELISA values of the serosal fluid cant differences were separated using Fisher’s pro- (dependent variable) vs time (independent variable) tected LSD. Following verification of differences for using linear and quadratic models using the PROC alkaloid transport using analysis of variance, means REG subroutine of SAS (SAS Inst. Inc.). for the serosal data for each tissue type (dependent Experiment 3: Transport of Ergot Alkaloids Across variable) were regressed against time (independent Ruminal and Omasal Tissues Treated with Sodium variable) using linear and quadratic models of the Azide. As in the first two experiments, digestive tissues PROC REG subroutine of SAS (SAS Inst. Inc.). from two ewes were used, and each ewe served as a tissue donor on a separate day. Four parabiotic cham- Results bers were prepared for each tissue from each animal. Two chambers for each tissue type were assigned ran- Experiment 1: Ergot Alkaloid Transport in Ruminal domly to one of two water baths. One of the two cham- and Omasal Tissues. Relative areas of the anterior dor- ber types within each water bath was randomly se- sal, posterior dorsal, anterior ventral, and posterior lected and 10 mL of KRPG containing a total of 30.5 ventral sacs of the rumen were similar (Table 1). The mmol each of lysergic acid, lysergol, ergonovine, ergo- relative area of the omasal tissue was greater than cryptine, and ergotamine was placed into the mucosal that of individual ruminal tissues but less than the chamber. The other chamber with the same tissue type sum of all ruminal tissues. Relative areas of the omasal within the waterbath had the same treatment applied, and ruminal tissues were greater than that of reticular but the KRPG contained 0.2% sodium azide to kill the tissue. Ruminal, omasal, and reticular tissues ac- tissues. Serosal chambers were sampled at 0, 60, 120, counted for approximately 53, 38, and 9% of the rela- 180, and 240 min after administration of the alkaloids tive surface area, respectively. to the mucosal chamber by aspirating 300 ␮L of solu- There were no tissue × alkaloid or time × alkaloid tion. Alkaloid appearance in the serosal chamber was treatment interactions for the β-hydroxybutyrate con- determined using the ELISA procedure as described centrations in the serosal fluid for this experiment (P by Adcock et al. (1997). β-Hydroxybutyric acid was > 0.05). Final serosal concentration of β-hydroxybutyr-

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Table 2. Final concentration of β-hydroxybutyrate in Table 3. Final serosal concentrations of individual serosal fluid of sheep digestive tissues incubated ergot alkaloids and potential alkaloid transport in parabiotic chambers for 4 h as measured by HPLC

Tissue type Serosal β-hydroxybutyrate, mg/dL Concentration Potential mole transport

Rumen anterior ventral 0.770 Ergot alkaloid Omasal Ruminal Omasal Ruminal Omasum 0.520b RMSE 0.090 ng/mL mmol Lysergic acid 993a 1,124a 26.6a 41.8a a,b < Means without a common superscript letter differ (P 0.05). Lysergol 442bc 657b 12.3b 25.4b Ergonovine 297c 260c 6. 7.6c Ergotamine 668b 398bc 6.9c 5.8c ate was greatest for ruminal and least for omasal tis- Ergocryptine 621b 260c 8.1bc 4.5c sue (Table 2). Healthy tissues were maintained in the Total 3,021 2,699 60.1 85.1 parabiotic chambers over the duration of time that RMSE 118 121 3.3 6.8 tissues were incubated because serosal β-hydroxybu- a,b,cColumn means without a common superscript letter differ (P < tyrate increased over time. Data for the other experi- 0.05). ments were similar (data not shown). Ergot alkaloid concentration in the serosal cham- tested. Serosal lysergic acid and ergonovine concentra- bers was affected by tissue type, time of incubation, tions did not differ among the tissues, but serosal er- and alkaloid treatment (alkaloid vs no alkaloid). There gotamine and ergocryptine were higher in the omasal was a tissue type × alkaloid × time interaction (P < tissue than in the ruminal tissue. Serosal lysergol con- 0.05). Initially, the serosal alkaloid values were simi- centration was greater when ruminal tissue was lar among tissue types, but ruminal tissue had more tested. alkaloid transported to the serosal chamber than did A tissue type × ergot alkaloid species interaction omasal tissue as time progressed (Figure 1). The re- occurred when potential alkaloid transport was calcu- gression equations best describing the tissue transport lated (P < 0.05). Lysergic acid had the highest potential of total alkaloids as measured by ELISA were qua- transport, regardless of tissue type, but was greater dratic for both tissues and had excellent fit to the data in ruminal than in omasal tissue (Table 3). Potential as demonstrated by the high coefficient of variation 2 transport of lysergol was equal to that of ergocryptine (r ). but greater than that of ergonovine or ergotamine in The concentration of the alkaloid species in the sero- omasal tissue. Lysergol had higher potential transport sal chambers was dependent on the type of tissue and than ergonovine, ergotamine, or ergocryptine in rumi- < alkaloid species (P 0.05). Of the alkaloids tested, nal tissue. Ergonovine, ergotamine, and ergocryptine lysergic acid had the greatest concentration in the se- had similar potential transport for both tissue types, rosal chambers regardless of tissue type (Table 3). The but potential lysergic acid and lysergol transport was serosal concentration of lysergol, ergotamine, and er- greater for ruminal tissue. gocryptine were not different, and ergonovine had the Experiment 2: Ergot Alkaloid Transport Across Rumi- lowest serosal concentration when omasal tissue was nal and Reticular Tissues. The ELISA values for alka- loid transport across ruminal digestive tissues were not different (P > 0.05). A quadratic equation gave the best fit to the data when the mean values from all ruminal tissue types were regressed with time (Figure 2). The regression equation had a good fit with the data, as was evidenced by the high simple coefficient of determination (r2 = 0.99). Potential alkaloid transport was greatest in the ru- minal posterior dorsal chamber for all alkaloids mea- sured (Table 4). As in Exp. 1, lysergic acid accounted for the greatest proportion of potentially transportable alkaloids for all tissues. There were no differences among reticular tissues for potentially transported lysergol, ergonovine, ergotamine, or ergocryptine, but the ruminal tissues had greater affinity for ergonovine transport than for lysergol. Ergotamine and ergocryp- tine had the least affinity for alkaloid transport among Figure 1. Ergot alkaloid transport across ruminal and all tissues. The reticular tissues transported the least omasal tissues as measured by enzyme-linked immuno- amount of alkaloids. sorbent assay over time. Alkaloid values represent nano- Experiment 3: Transport of Ergot Alkaloids Across grams per milliliter in the serosal fluid. Digestive Tissues Treated with Sodium Azide. Serosal

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Figure 2. Pooled reticular and ruminal data for ergot Figure 3. Effect of sodium azide on in vitro transport alkaloid transport as measured by enzyme-linked immu- of ergot alkaloids across omasal tissues. Alkaloid values nosorbent assay over time. Alkaloid values represent na- represent nanograms per milliliter in the serosal fluid. nograms per milliliter in the serosal fluid.

fescue toxicosis (Oliver et al., 1994; Moubarak et al., alkaloid concentrations as measured by ELISA were 1996). Recovery in those studies was only 1.25 to 12.5% similar among tissues treated with sodium azide for of the administered ergot alkaloids following intrave- the first 3 h of the experiment. At 4 h, the alkaloid nous injections at pharmacological concentrations (14 concentration in the serosal chamber of tissues treated ␮g/kg BW). Ergonovine was not detectable in serum with sodium azide was less than that of tissues that when it was continuously infused at “physiological con- received no azide and had little or no change from the centrations” and was present at concentrations of 1 to 3-h concentrations. A typical azide response is pre- 3 ng/mL serum when infused at four times an antici- sented in Figure 3. Mean values for β-hydroxybutyrate pated physiological rate for livestock grazing endo- were 0.38 and 0.13 mg/dL for tissues without and with phyte-infected tall fescue (Oliver et al., 1994). Low sodium azide treatments, respectively. Lower β-hydro- gastrointestinal absorption (Meier and Schreier, 1976; xybutyrate values for those treatments receiving so- Little et al., 1982) and rapid metabolism and clearance dium azide suggests that the tissue had died. Qua- of ergot alkaloids from the circulatory system make dratic regression equations gave the best fit to data the use of serum alkaloids difficult for investigation when sodium azide was omitted from the KRPG solu- of fescue toxicosis. Another approach is to examine tions, but linear equations gave best fits to the data tissue-specific alkaloid absorption. Parabiotic cham- when azide was added to the KRPG surrounding the bers have been used to investigate amino acid and tissues (Table 5). peptide transport in ruminal and omasal epithelial tissues (Matthews and Webb, 1995; Mathews et al., Discussion 1996a,b; McCollum and Webb, 1997, 1998). Our opin- ion is that parabiotic chambers have distinct advan- Researchers have attempted to use HPLC analysis tages over investigating in vivo absorption of ergot of serum to determine metabolism and pharmacoki- alkaloids because excised tissues are not subject to netics of ergot alkaloids in livestock suffering from associated in vivo complications with equilibrium dy-

Table 4. Mole equivalents of ergot alkaloids transported to the serosa of parabiotic chambers when equimolar concentrations were added in the mucosal chamber

Alkaloid Tissue Lysergic type Chamber Location Total acid Lysergol Ergonovine Ergotamine Ergocryptine

Potential transport, nmol Reticular Dorsal — 1,556c‡ 807c 337b 290b 61cd 71cd Reticular Ventral — 1,407c 897c 215b 244b 25d 26d Rumen Dorsal Posterior 5,925a 3,441a 578a 1,163a 327a 443a Rumen Dorsal Anterior 3,817b 2,685ab 318b 622b 83bcd 106bcd Rumen Ventral Posterior 3,733b 2,395ab 327b 633b 149ab 229b Rumen Ventral Anterior 3,318b 2,145b 305b 622b 129abc 180bc RMSE 613 784 135 289 55 95 a,b,c,dColumn means without a common superscript letter differ (P < 0.05).

Downloaded from jas.fass.org at USDA Natl Agricultural Library on December 17, 2008. Ergot alkaloid transport across tissues 547 Table 5. Linear and quadratic effects of time on appearance of ergot alkaloids in serosal fluids when various digestive tissues were incubated for 4 h with or without sodium azide

Linear Quadratic Tissue treatment Intercept coefficient coefficient r2

Rumen, + azide 0.26 (5.02)a 0.15 (0.03) 0.84 Rumen, no azide −0.52 (1.03) 0.06 (0.02) 0.0007 (0.0001) 0.99 Omasum, + azide 0.29 (3.13) 0.12 (0.06) 0.89 Omasum, no azide 4.45 (9.94) −0.39 (0.20) 0.0050 (0.0007) 0.98 aValues in parentheses represent standard deviations of the estimates of intercepts and linear and qua- dratic coefficients. Linear and quadratic coefficients were significantly different from 0.0 (P < 0.05). Intercepts were not different from 0.0. namics of receptors, compartmentalization of re- ganisms in antibody media used in our laboratory. It sponses within the body, and dynamics of the meta- is likely these concentrations were insufficient to cause bolic and excretory functions. immediate death of the tissues, because it took a mini- Ergot alkaloid transport to the serosal chamber oc- mum of 240 min to affect alkaloid transport. Reduced curred regardless of tissue type tested, but rates dif- β-hyroxybutyrate production and alkaloid transport fered among tissues. These potentially transported by tissues receiving the sodium azide treatment sug- quantities may not be indicative of in vivo transport gest that living tissue is necessary for transport to across digestive tissues, which is likely to be affected occur; hence, alkaloid transport is an active process. by retention time of the digesta within organs for each This suggests that feed additives that serve as compet- tissue type, the extent to which the liquid matrix inter- itors to receptors responsible for absorption could in- faces with the tissue surface, and the concentration of hibit alkaloid absorption. alkaloids within each digestive compartment. A crude Ergovaline has been ascribed the distinction of being approximation of alkaloid transport was calculated for the candidate toxin because of its association with en- tissues using serosal ergot alkaloid values. Ruminal dophyte-infected grasses (Rottinghaus et al., 1991; tissues varied in alkaloid transport and species of alka- Agee and Hill, 1994; Lane et al., 1997a,b) without the loid transported. Potential alkaloid transport across appropriate toxicological or physiological studies to reticular/ruminal and omasal tissues was calculated support the hypothesis. Piper et al. (1997) found little only at the 240-min period. These data suggest that effect of ergovaline on rats’ feed intake, weight gain, the rumen is capable of transporting approximately or serum prolactin when it was mixed in diets of endo- 25% more alkaloids than the omasum (Table 3) and phyte-free seed, but diets of endophyte-infected seed approximately 600% as much as the reticular tissues without added ergovaline decreased all these measure- (Table 4). Stuedemann et al. (1998) found that ergot ments. This suggests that alkaloids other than ergova- alkaloids were excreted in the urine within 12 h of line have a significant role in fescue toxicosis. The switching steers from endophyte-free to endophyte- results herein are paradoxical to the hypothesis that infected tall fescue pastures. They postulated that er- ergovaline (an ergopeptine alkaloid) is the toxin be- got alkaloids must be absorbed in the forestomachs of cause the simpler ergoline alkaloids have greater po- ruminants (i.e., reticular, ruminal, and omasal tis- tential transport. There is little doubt that the toxin(s) sues) because they are excreted rapidly. Data pre- is an ergot alkaloid because 1) monoclonal antibodies sented here support this hypothesis that the reticu- specific to the ergot alkaloids reverse fescue toxicosis lum, rumen, and omasum are absorptive sites of in- (Hill et al., 1994), 2) urinary alkaloid excretion of ergot gested ergot alkaloids. Stuedemann et al. (1998) also alkaloids is inversely proportional to average daily hypothesized that the metabolized alkaloids were er- gain of cattle (Hill et al., 2000), and 3) tall fescue goline alkaloids rather than ergopeptine alkaloids be- pastures containing novel endophytes without ergot cause of urinary excretion. In this experiment, the alkaloids result in superior animal performance (Bou- affinity of alkaloid transport for ergoline and ergopep- ton, 1998). However, little attention has been given tine alkaloids was tested. Regardless of tissue type, to the various forms of ergot alkaloids in endophyte- the ergoline alkaloids had a higher molar transport infected tall fescue or their ability to cross digestive than did the ergopeptine alkaloids (Tables 3 and 4), barriers, which is necessary for development of the thus confirming the hypothesis by Stuedemann et al. toxicosis syndrome. Moyer et al. (1993) found soluble (1998). However, higher transport of the simple ergo- ergovaline to be metabolized within ruminal fluid. line alkaloids is contrary to the common belief that Conversely, Stuedemann et al. (1998) determined that ergovaline (an ergopeptine alkaloid) is the toxic com- ergot alkaloids associated with the particulate fraction ponent. of ruminal fermentations became soluble and aggre- The concentration of sodium azide used to kill tis- gated in the liquid fraction. Both studies failed to iden- sues in Exp. 3 was similar to that used to kill microor- tify possible metabolites of the fermentation process.

Downloaded from jas.fass.org at USDA Natl Agricultural Library on December 17, 2008. 548 Hill et al. Results presented herein suggest that the simple Hill, N. S., F. N. Thompson, D. L. Dawe, and J. A. Stuedemann. ergoline alkaloids are more likely to cross digestive 1994. Antibody binding of circulating ergot alkaloids in cattle grazing tall fescue. Am. J. Vet. Res. 55:419–424. barriers than the ergopeptine alkaloids (Tables 3 and Hill, N. S., F. N. Thompson, J. A. Stuedemann, D. L. Dawe, and E. 4) and that the transport mechanism is an active pro- E. Hiatt, III. 2000. Urinary alkaloids as a diagnostic tool for cess. Identification and characterization of alkaloid re- fescue toxicosis in cattle. J. Vet. Diagn. Invest. 12:210–217. ceptors in the digestive tissue will be necessary to Lane, G. A., O. J. P. Ball, E. Davies, and C. Davidson. 1997a. 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