**4. Color Doppler ultrasonography in measuring vasoconstriction**

Color Doppler ultrasonography was introduced in the 1980s, but it was not until the 1990s that B-mode (two dimensional, gray scale) and color Doppler scans were combined as duplex images (Fig. 1) and used as a diagnostic or research tools in evaluating hemodynamics and increasing our knowledge of the characteristics and physiology of normal and abnormal blood flows. Color Doppler ultrasonography was used to evaluate hemodynamics and cross-sectional luminal areas of the cerebral artery in humans during

Fig. 1. A duplex image with B-mode image of a longitudinal section of the caudal artery of a heifer combined with color Doppler to exhibit blood flow.

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

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

**4. Color Doppler ultrasonography in measuring vasoconstriction** 

Color Doppler ultrasonography was introduced in the 1980s, but it was not until the 1990s that B-mode (two dimensional, gray scale) and color Doppler scans were combined as duplex images (Fig. 1) and used as a diagnostic or research tools in evaluating hemodynamics and increasing our knowledge of the characteristics and physiology of normal and abnormal blood flows. Color Doppler ultrasonography was used to evaluate hemodynamics and cross-sectional luminal areas of the cerebral artery in humans during

Fig. 1. A duplex image with B-mode image of a longitudinal section of the caudal artery of a

heifer combined with color Doppler to exhibit blood flow.

hyperplasia and not hypertrophy.

cattle and sheep exposed to ergot alkaloids.

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 the real-time color Doppler images.

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 spectra.

Doppler Ultrasonography

for Evaluating Vascular Responses to Ergopeptine Alkaloids in Livestock 573

Fig. 3. Duplex image of a longitudinal scan of the median caudal artery of a heifer showing

Determinations of constriction and blood flows in small arteries or veins are possible with Doppler ultrasonography if comparisons are made between with and without toxicant treatment groups of animals, or between baseline measures when animals are without toxicant exposure versus measures after exposure to toxicants. Walls of small veins possess smooth muscle and endothelium at the vessel intima that contain biogenic amide receptors; therefore, veins will elicit a vasoconstrictrive response to ergot alkaloids and facilitate a comparison between animals with and without exposure to ergot alkaloids. Unfortunately, elasticity of veins negates obtaining measures of blood flow resistance, which is valuable information on down-flow resistance. Small arteries possess thicker walls and layers of smooth muscle and are positioned upstream from capillary beds, which facilitate measurement of blood flow resistance, a relevant blood flow characteristic. Aiken et al. (2011) did not observe constriction in the carotid arteries of lambs over a 9-d period of grazing perennial ryegrass infected with a novel endophyte that produced ergovaline, but the carotid showed linear increases in pulsatility, a measure of blood flow resistance (Petersen et al., 1997). It was concluded that constriction of cranial arteries were the source

Emphasis on blood flow to peripheral tissues facilitates use of high frequency (≥ 5 MHz) transducers that will provide high resolution imaging of vessels at low depths from the transducer. Hemodynamics can be studied for vessels less than 2 to 3 cm from the skin surface and supplying blood to the tissues of the extremities. Cross-sectional images of the medial caudal artery (Fig. 4) of cattle has been imaged at the base of the tail to evaluate constrictive responses of the artery to ergot alkaloids (Aiken et al., 2007; 2009b; Kirch et al., 2008). For sheep, flow through the caudal artery is affected from the docking of tails and of questionable reliability, but cross-sectional images of the posterior auricular artery (Fig. 5) that supplies blood to the ear can reliably be used for assessing vasoconstriction ( Aiken et al., 2011). Cross-sections of the medial palmer artery of the forelimb also have been imaged (Fig. 6; unpublished data collect by K. McDowell; Dept. of Veterinary

the sample volume cursor (upper image) and the Doppler spectra (lower image).

**4.1 Doppler imaging the vasculature of livestock** 

of resistance detected in the carotid arteries.

Sciences; University of Kentucky).

Fig. 2. Transmission (ft) and receiving (fr) ultrasound beams for the detection of Doppler signals from blood flow. The Doppler angle, θ, is the angle between the directions of blood flow and the beam. Space within the dashed lines represents the sample volume (Adapted from Zagzebski, 2000).

Blood flow velocity is determined by calculating Doppler Frequency (ƒD):

$$f\_{\rm D} = 2f\_{\rm t} \text{v} \cos \theta / \text{c}$$

where ƒt is the transmitted frequency, ƒr is the received frequency, v is the blood flow velocity, θ is the Doppler angle between the axis of the ultrasound beam and the direction of flow , and c is the speed of sound. The Doppler effect allows the calculation of velocity of a moving object as the frequency of sound waves generated by the object increases as it moves from a far to a near distance. Calculation of blood flow velocity is not possible if the ultrasound beam is parallel with blood flow (i.e., θ = 90°). Angling of the ultrasonic bean must be done through angling of the transducer or using the beam steering with phased array transducers.

Pulse-wave transducers are designed to receive frequencies of scattered sound waves bounced from moving particles (e.g., hemoglobin) within the sample volume cursor of the targeted vessel (Fig. 3). Cross-sectional color Doppler images can be used to trace luminal areas of vessels and combined with blood flow velocities derived from Doppler spectra from a longitudinal image of the vessel to calculate blood flow rate (mean velocity X cross-sectional luminal area). Luminal areas determined from cross-sectional scans of vessels can be used to evaluate contractile responses of smooth muscle within artery and vein walls to environmental conditions, pharmaceuticals, or toxicants. Therefore, duplex imaging of B-mode and color Doppler ultrasound images has provided a tool for conducting either diagnoses or scientific research in real time that is noninvasive, and can be repeatable and objective by following good scanning technique and interpretation procedures.

Fig. 2. Transmission (ft) and receiving (fr) ultrasound beams for the detection of Doppler signals from blood flow. The Doppler angle, θ, is the angle between the directions of blood flow and the beam. Space within the dashed lines represents the sample volume (Adapted

ƒD = 2ƒtvcosθ/c where ƒt is the transmitted frequency, ƒr is the received frequency, v is the blood flow velocity, θ is the Doppler angle between the axis of the ultrasound beam and the direction of flow , and c is the speed of sound. The Doppler effect allows the calculation of velocity of a moving object as the frequency of sound waves generated by the object increases as it moves from a far to a near distance. Calculation of blood flow velocity is not possible if the ultrasound beam is parallel with blood flow (i.e., θ = 90°). Angling of the ultrasonic bean must be done through angling of the transducer or using the beam steering with phased

Pulse-wave transducers are designed to receive frequencies of scattered sound waves bounced from moving particles (e.g., hemoglobin) within the sample volume cursor of the targeted vessel (Fig. 3). Cross-sectional color Doppler images can be used to trace luminal areas of vessels and combined with blood flow velocities derived from Doppler spectra from a longitudinal image of the vessel to calculate blood flow rate (mean velocity X cross-sectional luminal area). Luminal areas determined from cross-sectional scans of vessels can be used to evaluate contractile responses of smooth muscle within artery and vein walls to environmental conditions, pharmaceuticals, or toxicants. Therefore, duplex imaging of B-mode and color Doppler ultrasound images has provided a tool for conducting either diagnoses or scientific research in real time that is noninvasive, and can be repeatable and objective by following good scanning technique and interpretation

Blood flow velocity is determined by calculating Doppler Frequency (ƒD):

from Zagzebski, 2000).

array transducers.

procedures.

Fig. 3. Duplex image of a longitudinal scan of the median caudal artery of a heifer showing the sample volume cursor (upper image) and the Doppler spectra (lower image).

#### **4.1 Doppler imaging the vasculature of livestock**

Determinations of constriction and blood flows in small arteries or veins are possible with Doppler ultrasonography if comparisons are made between with and without toxicant treatment groups of animals, or between baseline measures when animals are without toxicant exposure versus measures after exposure to toxicants. Walls of small veins possess smooth muscle and endothelium at the vessel intima that contain biogenic amide receptors; therefore, veins will elicit a vasoconstrictrive response to ergot alkaloids and facilitate a comparison between animals with and without exposure to ergot alkaloids. Unfortunately, elasticity of veins negates obtaining measures of blood flow resistance, which is valuable information on down-flow resistance. Small arteries possess thicker walls and layers of smooth muscle and are positioned upstream from capillary beds, which facilitate measurement of blood flow resistance, a relevant blood flow characteristic. Aiken et al. (2011) did not observe constriction in the carotid arteries of lambs over a 9-d period of grazing perennial ryegrass infected with a novel endophyte that produced ergovaline, but the carotid showed linear increases in pulsatility, a measure of blood flow resistance (Petersen et al., 1997). It was concluded that constriction of cranial arteries were the source of resistance detected in the carotid arteries.

Emphasis on blood flow to peripheral tissues facilitates use of high frequency (≥ 5 MHz) transducers that will provide high resolution imaging of vessels at low depths from the transducer. Hemodynamics can be studied for vessels less than 2 to 3 cm from the skin surface and supplying blood to the tissues of the extremities. Cross-sectional images of the medial caudal artery (Fig. 4) of cattle has been imaged at the base of the tail to evaluate constrictive responses of the artery to ergot alkaloids (Aiken et al., 2007; 2009b; Kirch et al., 2008). For sheep, flow through the caudal artery is affected from the docking of tails and of questionable reliability, but cross-sectional images of the posterior auricular artery (Fig. 5) that supplies blood to the ear can reliably be used for assessing vasoconstriction ( Aiken et al., 2011). Cross-sections of the medial palmer artery of the forelimb also have been imaged (Fig. 6; unpublished data collect by K. McDowell; Dept. of Veterinary Sciences; University of Kentucky).

Doppler Ultrasonography

for Evaluating Vascular Responses to Ergopeptine Alkaloids in Livestock 575

Other vessels can be scanned, but decisions of which vessels to study in livestock animals should be partly based on ease and safety for a particular image site. Imaging of larger livestock that must be handled in chutes (i.e., pastured animals with minimal exposure to humans) or stanchions should be restricted to exposed upper regions. Ultrasounding vessels in legs should be restricted to haltered animals at ease with human contact because of the vulnerability to operator injury and equipment damage. Major arteries (e.g., aorta, carotid, mysenteric, etc.) and those supplying blood to major organs (e.g., hepatic, pulmonary, cerebral, etc.) can be studied to ascertain toxicant effects on organ function. These evaluations at greater tissue depths must be done with low pulse frequencies (2 to 5 Mhz) to obtain the needed resolution and with high end, high resolution ultrasound units. It is further recommended these studies be done with haltered, calm animals in controlled environments. Further, obese animals should be avoided because thick layers of subcutaneous adipose will cause interference in achieving high resolution with B-mode

Lumen areas of vessels can be traced from B-mode scans with or without color Doppler imaging (Fig. 7). Aiken et al. (2009a) concluded that artery luminal areas can be measured with similar precision by tracing the intima of connective tissue in the artery wall or the outer boundary of the color Doppler flow signal. Color Doppler imaging for measuring luminal area will require images depicting the maximum flow signal which will coincide with peak systole. These images can be identified using the cine memory of the ultrasound unit. There are two methods of imaging color blood flow. Color Doppler uses the Doppler frequency shift to detect flow within the vessel and shifts the color shade from dark to light as frequency and velocity increases. Power Doppler imaging uses the power or intensity of the Doppler signal to show differences in blood flow velocities within a vessel. Power Doppler is generally more sensitive than color Doppler in detecting blood flow. Without color Doppler, luminal areas should be at certain points of a cardiac cycle (e.g., peak systole or end diastole) identified using Doppler spectra. The ultrasound beam should be perpendicular to blood flow when measuring cross-sectional luminal areas, as deviations from perpendicular will enlarge luminal areas. A 10° departure from perpendicular will increase luminal area by approximately 5%. Lumen area diameter also can be measured from longitudinal images of vessels, but the ultrasonic beam must be centered over the vessel. Departures from this center point will

As previously discussed, Doppler spectra are derived from blood flow within the sample volume cursor or gate, which can be adjusted to various sizes. A small sample volume can be used and positioned in the middle of the vessel, which has the highest velocity with luminar blood flow; however, inconsistent positioning with this approach can inflate between image variations. A wider sample volume can provides a measure of mean flow velocity within the vessel and result in less between-image variability. The sample volume should be consistent width between animals in a study to reduce this source of

Aliasing of color displays and Doppler spectra can occur when using pulse Doppler. Aliasing is caused by a wraparound of the display, with high velocity flow moving towards

Doppler images taken at low frequencies (Pozniak, 2000).

be biased towards smaller luminal areas.

variation.

Fig. 4. Cross-sectional image of the medial caudal artery in a heifer calf.

Fig. 5. Cross-sectional image of the right auricular artery of a ewe lamb.

Fig. 6. Cross-sectional image of the medial palmer artery of a mare.

Fig. 4. Cross-sectional image of the medial caudal artery in a heifer calf.

Fig. 5. Cross-sectional image of the right auricular artery of a ewe lamb.

Fig. 6. Cross-sectional image of the medial palmer artery of a mare.

Other vessels can be scanned, but decisions of which vessels to study in livestock animals should be partly based on ease and safety for a particular image site. Imaging of larger livestock that must be handled in chutes (i.e., pastured animals with minimal exposure to humans) or stanchions should be restricted to exposed upper regions. Ultrasounding vessels in legs should be restricted to haltered animals at ease with human contact because of the vulnerability to operator injury and equipment damage. Major arteries (e.g., aorta, carotid, mysenteric, etc.) and those supplying blood to major organs (e.g., hepatic, pulmonary, cerebral, etc.) can be studied to ascertain toxicant effects on organ function. These evaluations at greater tissue depths must be done with low pulse frequencies (2 to 5 Mhz) to obtain the needed resolution and with high end, high resolution ultrasound units. It is further recommended these studies be done with haltered, calm animals in controlled environments. Further, obese animals should be avoided because thick layers of subcutaneous adipose will cause interference in achieving high resolution with B-mode Doppler images taken at low frequencies (Pozniak, 2000).

Lumen areas of vessels can be traced from B-mode scans with or without color Doppler imaging (Fig. 7). Aiken et al. (2009a) concluded that artery luminal areas can be measured with similar precision by tracing the intima of connective tissue in the artery wall or the outer boundary of the color Doppler flow signal. Color Doppler imaging for measuring luminal area will require images depicting the maximum flow signal which will coincide with peak systole. These images can be identified using the cine memory of the ultrasound unit. There are two methods of imaging color blood flow. Color Doppler uses the Doppler frequency shift to detect flow within the vessel and shifts the color shade from dark to light as frequency and velocity increases. Power Doppler imaging uses the power or intensity of the Doppler signal to show differences in blood flow velocities within a vessel. Power Doppler is generally more sensitive than color Doppler in detecting blood flow. Without color Doppler, luminal areas should be at certain points of a cardiac cycle (e.g., peak systole or end diastole) identified using Doppler spectra. The ultrasound beam should be perpendicular to blood flow when measuring cross-sectional luminal areas, as deviations from perpendicular will enlarge luminal areas. A 10° departure from perpendicular will increase luminal area by approximately 5%. Lumen area diameter also can be measured from longitudinal images of vessels, but the ultrasonic beam must be centered over the vessel. Departures from this center point will be biased towards smaller luminal areas.

As previously discussed, Doppler spectra are derived from blood flow within the sample volume cursor or gate, which can be adjusted to various sizes. A small sample volume can be used and positioned in the middle of the vessel, which has the highest velocity with luminar blood flow; however, inconsistent positioning with this approach can inflate between image variations. A wider sample volume can provides a measure of mean flow velocity within the vessel and result in less between-image variability. The sample volume should be consistent width between animals in a study to reduce this source of variation.

Aliasing of color displays and Doppler spectra can occur when using pulse Doppler. Aliasing is caused by a wraparound of the display, with high velocity flow moving towards

Doppler Ultrasonography

for Evaluating Vascular Responses to Ergopeptine Alkaloids in Livestock 577

Fig. 8. Aliasing of the flow display with a color Doppler image

**4.2 Using serial images to determine sensitivities and response to toxicants** 

Direct comparisons in luminal vessel areas and blood flow characteristics can be made with color Doppler ultrasonography between groups of animals that are with or without exposure to toxicants. Of greater importance, however, can be in measuring vascular sensitivity to toxicants. The research tool has successfully measured vascular sensitivity to ergot alkaloids in cattle (Aiken et al. 2007; 2009b) and sheep (Aiken et al., 2011), and to determine vascular recovery of sheep after being switched from toxic endophyte-infected pastures of perennial ryegrass to endophyte-free ryegrass (Aiken et al., 2011). Following an initial feeding of a diet containing 0.8 mg ergovaline/kg dry matter to heifers, Aiken et al. (2007) observed a tendency for a vasoconstrictive response by the caudal artery in 4 hours. A similar experiment that fed treatment diet concentrations of 0, 0.2, and 0.8 mg ergovaline/kg dry matter to heifers determined there were vasoconstrictive responses by the caudal artery in 27 hours for the 0.8 mg ergovaline/kg dry matter diet concentration, and in 51 hours for

Fig. 9. Aliasing of the Doppler spectra.

the 0.4 mg ergovaline/kg DM dry matter.

(a)

(b)

Fig. 7. Cross-sectional B-mode images of the medial caudal artery (vessel on right) and vein (vessel on left) in a heifer calf, without (a) or with (b) color Doppler.

the probe (i.e., expected to be red) appearing that is moving away from the probe (Fig. 8; i.e., displayed to be blue). This aliasing can be eliminated by increasing the pulse repetition frequency, which increases the color flow velocity scale. Aliasing also occurs with Doppler spectra, with the spectra of high velocity blood flow being converted to reverse flow spectra (Fig. 9). As with the color display, the pulse repetition frequency can be increased to increase the flow velocity scale. The operator also can reduce the spectra baseline down until the spectra represents flow in one direction.

(a)

(b)

Fig. 7. Cross-sectional B-mode images of the medial caudal artery (vessel on right) and vein

the probe (i.e., expected to be red) appearing that is moving away from the probe (Fig. 8; i.e., displayed to be blue). This aliasing can be eliminated by increasing the pulse repetition frequency, which increases the color flow velocity scale. Aliasing also occurs with Doppler spectra, with the spectra of high velocity blood flow being converted to reverse flow spectra (Fig. 9). As with the color display, the pulse repetition frequency can be increased to increase the flow velocity scale. The operator also can reduce the spectra baseline down until the

(vessel on left) in a heifer calf, without (a) or with (b) color Doppler.

spectra represents flow in one direction.

Fig. 8. Aliasing of the flow display with a color Doppler image
