**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 the 0.4 mg ergovaline/kg DM dry matter.

Doppler Ultrasonography

vasculature.

after imaging.

**4.3 Special considerations in animal handling** 

handling procedures are followed.

for Evaluating Vascular Responses to Ergopeptine Alkaloids in Livestock 579

ryegrass to ryegrass infected with a novel endophyte that produces high concentrations of ergovaline. A linear decline in luminal area of the auricular artery was observed over a 9 day after grazing was initiated. Lambs also were switched from AR6 novel endophyte ryegrass to endophyte-free pasture. Over the 9 days, luminal area of the auricular artery increased linearly and pulsatillity indices for the carotid decreased linearly. There was relaxation of the vasculature from alkaloid clearance, but a longer duration of data collection was needed to determine the time needed for complete clearance of alkaloids from the

Accuracy and precision of measurements of blood flow characteristics in livestock are vastly improved if the measurements are collected in controlled environments with halter broke animals that are at ease with human contact. Furthermore, controlled diets fed in pens with know concentrations of toxicants will be necessary if research objectives are focused on determining sensitivities and strength of response to the toxicant. Blood flow measurements for pastured animals with minimal contact with humans will be affected by animal nervousness (e.g. increases in heart rate). However, measurements from ultrasound images are possible in uncontrolled environments and without halter-broke animals if control animals (e.g., animals grazing toxicant-free pastures) are measured and special animal

Pastured cattle are typically handled in squeeze chutes with narrow alleys (Fig. 11) leading to the chute. Cattle are gregarious and individuals become nervous when singly separated from the group. When imaging cattle in squeeze chutes, the time individuals spend in the alley prior to being imaged should be minimized. Rather than crowding the alley, cattle should be placed in the alley in groups of three, with subsequent groups of three being placed in the alley such that an animal is not alone in the alley. Once in the chute, the sides of the chute should be reduced to not squeeze the animal to a point of discomfort, but reduce the area containing the animal. Potential nervousness of cattle worked in chutes makes it difficult to image cranial regions; therefore, imaging is better done if restricted to caudal regions (e.g., caudal artery or vein). The area should also be as quiet of sound, as possible, to minimize animal startling and raising of heart rates. Further, any sample collections (e.g., blood) or measurements (e.g., rectal or skin temperatures) should be done

Imaging of horses can typically be done in stanchions with those that are haltered and at ease with humans (Fig. 12). Sheep and goats tend to also be gregarious, but do not present the same degree of nervousness as cattle. Small ruminants can be worked on cradles (Fig. 13) and provide opportunities to reliably image the cranial vessels (Aiken et al., 2011).

Imaging animals in an uncontrolled environment requires that images be collected over a period of time during the day with minimal changes in air temperature and humidity (Kirch et al., 2008). Imaging quality also is a major consideration in providing reliable interpretations and measures. Time must be taken to correctly place tranducers over the vessels, with sufficient gain and focus settings to optimize B-mode and color Doppler images, and Doppler spectra, Therefore, time of day and time necessary to collect quality

Serial imaging also can be used to determine the extent and persistence of the vascular constriction response to toxicants. Following a reduction in luminal area and blood flow rate in heifers consuming diets consuming 0.4 or 0.8 mg ergovaline/kg DM, Aiken et al. (2007) reported adjustments in luminal area and blood flow velocity to original baseline measures, taken when heifers were on a 0 mg ergovaline/kg DM diet concentration adjustment diet, as air temperatures increased (Fig. 10). However, animals on control diets have shown greater variation in blood flow than those on treatment diets containing ergot alkaloids. Aiken et al. (2007; 2009b) concluded there was greater variation in caudal artery luminal areas and blood flow rates among control cattle compared to those on treatment diets (refer to standard error bars in Fig. 10), making it necessary to evaluate deviations from baseline measures for determine alkaloids effects. Low variation in caudal artery luminal areas among cattle on the treatment diet was suggested to be associated with persistent vasoconstriction causing an inability to adjust their vasculature to changes in ambient temperature.

Fig. 10. Trends in luminal area of the caudal artery (a) and blood flow rate through the caudal artery (b) in heifer calves flowing placement of cattle on experimental diets (0, 0.4, or 0.8 mg ergovaline/kg DM) (from Aiken et al., 2009b).

Aiken et al. (2011) used color Doppler ultrasound to determine constriction of the auricular and carotid arteries in ewe lambs after they were switched from endophyte-free perennial ryegrass to ryegrass infected with a novel endophyte that produces high concentrations of ergovaline. A linear decline in luminal area of the auricular artery was observed over a 9 day after grazing was initiated. Lambs also were switched from AR6 novel endophyte ryegrass to endophyte-free pasture. Over the 9 days, luminal area of the auricular artery increased linearly and pulsatillity indices for the carotid decreased linearly. There was relaxation of the vasculature from alkaloid clearance, but a longer duration of data collection was needed to determine the time needed for complete clearance of alkaloids from the vasculature.
