**3. Ergopeptnes and their effects on vascular circulation**

Ergopeptines are ergot alkaoids that are produced by an endophyte (*Neotyphodium coenphialum*) that infects tall fescue plants (Bacon, 1995), and by the *N. lolii* endophyte that infects perennial ryegrass (Easton & Tapper, 2005). Although the endophyte that infects perennial ryegrass produces small amounts of ergot alkaloids that can induce vasoconstriction (Aiken et al., 2011), it is its production of lolitrem B that causes ryegrass staggers cattle and sheep which is of greater concern (Fletcher and Harvey, 1981). Livestock exhibiting staggers are incapacitated due to tremors, but the malady also affects animal performance (Siegel et al., 1985). All plant parts of tall fescue and perennial ryegrass contain ergot alkaloids, but alkaloid concentrations differ among plant parts. Rottinghaus et al. (1991) determined the ranking of plant parts from highest to lowest ergot alkaloid concentrations is seed, stem, leaf sheath, and leaf blade. There is a distinction between the mutual relationships between the wild-type, toxic endophytes that infect naturalized populations of tall fescue and perennial ryegrass and non-ergot alkaloid producing novel endophytes that are artificially infected into commercially released cultivars of each grass (Bouton et al., 2002). Claviceps spp. also produce ergot alkaloids, but fungal colonization and alkaloid concentrations are restricted to the seed or grain (Bandyopadhyay et al., 1998).

Ergovaline has been proposed as the likely causal agent in the fescue toxicosis syndrome (Lyons et al, 1986). In vitro electromyograph studies have reported ergovaline to cause contractile responses of bovine uterine and umbilical arteries (Dyer, 1993), rat tail and guinea pig iliac arteries (Schoning, et al., 2001), and lateral saphenous weins of cattle (Klotz et al., 2007). Klotz et al. (2007) reported similar contractile responses between ergovaline and ergotamine, with contractile responses being initiated at 1 x 10-8 M concentrations for both ergopeptines. An earlier experiment by Klotz et al. (2006) determined a weak in vitro contractile response of the lateral saphenous vein to lysergic acid (a structurally simpler ergot alkaloid (reviewed by Strickland et al., 2011) that did not mediate contraction until concentrations reached supraphysiological levels (1 x 10-4 M). Dyer (1993) and Schoning et al. (2001) both showed that ergovaline elicited its contractile effects through activation of 5HT2A serotonergic receptors. However, in contrast to Dyer (1993) who showed that the 1-adrenergic receptor was not important in the contractile effects of ergovaline on the bovine uterine and umbilical arteries; Schoning et al. (2001) clearly demonstrated that the 1-adrenergic receptors were important to vascular regulation by ergovaline in their blood vessel models. Similar findings have been noted for other ergot alkaloids produced by both Neotyphodium and Claviceps spp. (reviewed by Strickland et al., 2009a, b; Strickland et al., 2011).

Although in vitro models are useful tools for investigating and identifying the modes by which the ergot alkaloids may effect vascular dysfunction, the data from these models must be interpreted with care until fully validated by in vivo models. Partial validation is provided by the results of earlier in vivo studies. Lewis and Gelfand (1935) demonstrated that ergotamine treatment of chickens resulted in cessation of blood flow to the comb and subsequent gangrene. They postulated that the gangrene developed as a result of endothelia damage. Shappell (2003) demonstrated that ergovaline was, in fact, directly cytotoxic to a

Doppler Ultrasonography

the real-time color Doppler images.

spectra.

for Evaluating Vascular Responses to Ergopeptine Alkaloids in Livestock 571

hypoxia and hypercapnia (Poulin and Robbins, 1996). Wendelhag et al. (1991) utilized Bmode ultrasonography to measure carotid luminal and plaque areas in humans. Doppler spectra have provided estimates of blood flow characteristics in arteries of the dog (Lee et al., 2004) and horses (Raisis et al., 2000; Hoffman et al., 2001). Recently, color doppler ultrasonography was performed in evaluating vasoconstriction and blood flow responses to ergot alkaloid exposure in cattle (Aiken et al. 2007; 2009b) and sheep (Aiken et al., 2011). Therefore, the technology has been utilized to acquire baseline measures of normal flood flow characteristics and assessing vascular constriction as a factor of abnormal blood flow. It is beyond the scope of this chapter to provide a detailed description of the mechanics and physics associated with color Doppler ultrasonography, but some basic fundamentals are necessary for understanding the procedures and potential errors when using the technology. Basically, the transducer transmits a frequency of ultrasonic sound waves (1 to 30 MHz) through tissues and, depending on the densities of the tissues the ultrasound beam contacts, a portion of the waves pass through a particular tissue and the remainder is reflected. Piezoelectric crystals in the transducer transmit and receive wave frequencies and the ultrasound unit compiles and converts the wave energy to electrical energy. There is a progressive loss of acoustic energy as ultrasonic beams passes through tissues, which is referred to as attenuation. Attenuation is the decrease in the intensity of returning sound waves as ultrasonic beams are transmitted into deeper tissues. Amount of attenuation, measured in dB/cm, is dependent on depth of ultrasonic beam, density of the tissue the beam passes through, and the frequency of the transmitted beam (Sites et al., 2007). Gain settings (i.e., separate settings for B-mode, color Doppler, and Doppler spectra) affect sensitivity. Amplitude and velocity information of the transmitted versus received frequencies are used to compute and delineate anatomical structures viewed in B-mode images, blood flow identification, and velocity within the sample volume cursor placed in

There are two types of transducers used in color Doppler ultrasonography, continuous- and pulse-wave Doppler transducers. Continuous-wave transducers use 2 crystal units to continuously transmit and receive ultrasound waves. They can accurately measure a wide range of flow velocities within a vessel, but lacks an ability to control location of measurements within the vessel lumen. Most Doppler ultrasound units use pulsed-wave transducers that alternate groups of crystals in transmitting and receiving ultrasound wave in pulses to provide Doppler shift data from a specified area within the vessel lumen. This specified area, referred to as the sample volume, is set by the operator. Ultrasonic beans also can be angled with curvilinear and linear phased array transducers. For pulse Doppler scanning, the same mechanisms apply as with B-mode scans, but the sample volume cursor is placed in a B-mode image of a longitudinally scanned vessel and within the color generated by blood flow (red for flow towards the transducer and blue for flow away from the transducer). Doppler spectra derived from blood flow within the sample volume cursor can be automatically or manually traced for a given cardiac cycle to measure systolic velocity, end diastolic velocity, mean velocities, and resistance and pulsatility indices using algorithms in the computer of most Doppler ultrasound units. Most Doppler ultrasound units also will provide measures of heart rate, time, and acceleration. The longitudinal color Doppler image and Doppler spectra are typically combined (Fig. 2), with the color Doppler scan being utilized for placement of the sample volume cursor for the observed Doppler

number of cell types, including endothelial cells. Additional morphological changes and blood flow dysfunction of peripheral tissues in cattle have been reported (Walls and Jacobson, 1970; Julien et al., 1974; Williams et al., 1975), including: blood vessel congestion, perivascular hemorrhage, blood vessel distension, and thickened blood vessel walls with small lumens. Strickland et al. (1996), provided evidence, using bovine vascular smooth muscle cells in vitro, that the thickened blood vessel walls were likely the result of hyperplasia and not hypertrophy.

In addition to the noted effects in the peripheral vascular beds, Rhodes et al. (1991) reported ergot alkaloid induced in vivo constrictive responses in several vascular beds of steers. Using radiolabeled microspheres, steers fed diets with high ergot alkaloid concentrations (2.6 mg egovaline/kg DM) had reduced blood flow to rib skin, cerebellum, duodenum, and colon, as compared to those fed low ergot alkaloid diets (< 0.05 mg ergovaline/kg DM). They also reported similar findings in sheep. The aforementioned in vivo studies have provided partial validation of the in vitro findings to date. Further, the in vitro data coupled with the in vivo findings support a strong role of the ergot alkaloids in vascular dysfunction. However, none of the aforementioned studies have been capable of studying, in real time, the effects of the ergot alkaloids on vascular function in the intact and unaltered animal. Color Doppler Ultrasonography offers the opportunity to do exactly that as is evidenced by recent publications (Aiken et al., 2007, 2009b, and 2011) concerning blood flow changes in cattle and sheep exposed to ergot alkaloids.
