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Avoidance of phenylthiocarbamide in mature Targhee and Rambouillet rams

Avoidance of phenylthiocarbamide in mature Targhee and Rambouillet rams Downloaded from https://academic.oup.com/tas/article-abstract/3/4/txz125/5581782 by Ed 'DeepDyve' Gillespie user on 18 October 2019 † ‡, ,1 Dillan Henslee,* Joel Yelich, J. Bret Taylor, and Melinda Ellison* *Department of Animal and Veterinary Science, University of Idaho, Moscow, ID 83844; Department of Animal and Veterinary Science, Idaho Agricultural Experiment Station, Nancy M. Cummings Research, Extension and Education Center, University of Idaho, Carmen, ID 83462; and U.S. Sheep Experiment Station, Agricultural Research Service, USDA, Dubois, ID 83423 ABSTRACT:  Shrub encroachment on grass- 2  h in trial 2.  Each test day was followed by a lands is a worldwide issue and sheep are a po- rest day where PTC solution was replaced with tential tool for mitigating shrub encroachment. water to limit potential carry over effects into Many shrubs, however, contain bitter-tasting the next test day. Consumption of PTC solution compounds that may deter grazers. Cattle and for each PTC concentration was expressed as sheep commonly graze rangelands, but of the the percentage of PTC solution intake of total two, sheep have a greater tolerance for bitter morning fluid intake. There was no effect (P > compounds and would be expected to consume 0.74) of sequence that rams received PTC solu- more bitter-tasting vegetation. We hypothesized tions on PTC consumption during either trial. that sheep could detect (i.e., taste) bitter-tasting As PTC concentration increased, percentage compounds and the sensitivity to these com- of PTC solution intake decreased (P ≤ 0.01) pounds would vary from animal to animal. The for both trials. The greatest decrease in per- objective of this study was to determine whether centage of PTC solution intake occurred be- sheep could detect the bitter-tasting compound tween 1.57 and 4.39  mM (58%) for trial 1 and phenylthiocarbamide (PTC), and if so, what 2.03 and 4.39  mM (72%) for trial 2.  In trial 2, PTC concentration would elicit an avoidance the least percentage of PTC solution intake was response. Using a crossover study design, ma- the 4.39  mM PTC concentration, which was ture Rambouillet and Targhee rams (n  =  30) different (P ≤ 0.05) from lesser PTC concen- were subjected in randomized order to various trations. All other PTC concentrations did not PTC concentrations mixed in the drinking water differ (P > 0.05) from each other in percentage (PTC solution). In trials 1 and 2 (n  =  15/trial), intake. This research suggests rams could taste 0.20, 0.56, 1.57, 4.39, and 12.29  mM and 0.20, the PTC, and the concentration at which PTC 0.43, 0.94, 2.03, and 4.39  mM of PTC were solution was avoided varied across rams. It may tested, respectively. On test days, PTC solution be possible to select sheep, based on demon- (trial 1: 1.5 kg; trial 2: 3.0 kg) and water (same strated avoidance of PTC, for targeted grazing amounts) were offered for ad libitum intake in applications to manipulate vegetation toward a side-by-side presentation for 1 h in trial 1 and range management goals. Key words: bitterness avoidance, phenylthiocarbamide, sagebrush, sheep © The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Transl. Anim. Sci. 2019.XX:0-0 doi: 10.1093/tas/txz125 Corresponding author: ellison@uidaho.edu Received April 18, 2019. Accepted August 7, 2019. 1 Downloaded from https://academic.oup.com/tas/article-abstract/3/4/txz125/5581782 by Ed 'DeepDyve' Gillespie user on 18 October 2019 2 Henslee et al. INTRODUCTION (h   =  0.55) (Morton et  al., 1981). It has also been suggested that PTC avoidance is influenced by post- Overgrowth of mountain big sagebrush digestive factors (Nelson et al., 2003), similar to the (Artemisia tridentate Nutt. ssp. vaseyana) can lead preferences of sheep grazing bitter/toxic forages to a reduction in rangeland plant diversity, carrying (Launchbaugh et al., 2001). capacity, and wildlife abundance (Johnson et  al., This study focused on bitter taste avoidance 1996; Launchbaugh, 2003). Sagebrush can be con- (Parker, 2003) by the addition of PTC in water. We trolled or eliminated by plowing, burning, and hypothesized that sheep could detect bitter-tasting spraying (Wambolt and Payne, 1986), but these compounds and the sensitivity would vary from methods can be expensive and have potential un- animal to animal. The objective of this study was desirable effects on rangelands. One method of to determine whether sheep could detect the bitter control that has received minimal attention, and tasting compound, phenylthiocarbamide, and if so, may be more sustainable, is reduction of sagebrush what PTC concentration would elicit an avoidance with grazing sheep. Using fecal analysis over sev- response. eral experiments, Snowder et  al. (2001) indicated that the dietary preference for sagebrush in sheep MATERIALS AND METHODS has a heritability of 0.28, suggesting that selection against bitterness avoidance in sheep breeding pro- Animals grams may be feasible. Furthermore, Ferreira et al. (2013) identified a set of novel genes for bitter taste All animal procedures were approved by an receptors in sheep, suggesting that sheep may be Institutional Animal Care and Use Committee genetically predisposed to select or avoid plants (USDA, ARS, Dubois, ID) in accordance with the with bitter or noxious tastes. USDA, APHIS Animal Welfare Regulations (2013; Sheep are adaptive selective grazers 9 C.F.R. § 2.30-2.38 2013) and the Guide for the Care (Launchbaugh et  al., 2001) with varying dietary and Use of Agricultural Animals in Research and preference for consuming sagebrush (Bork et  al., Teaching (FASS, 2010). Two trials were conducted 1998; Snowder et al., 2001; Seefeldt, 2005). Several at the USDA, ARS U.S. Sheep Experiment Station factors can be attributed to an individual’s diet pref- (USSES) located near Dubois, Idaho in the spring erence/selection including learned behaviors, taste of 2018. In trial 1, yearling Rambouillet (n = 7) and preference, postdigestive feedback, and their ability Targhee (n = 8) rams (initial BW = 76.6 ± 5.7 and to detoxify secondary metabolites. Many toxic for- 83.7 ± 9.1 kg, respectively) were used; while in trial ages have a bitter taste, but the toxicity and the cor- 2, yearling Rambouillet (n = 6) and Targhee (n = 9) relation of bitter taste to toxicity varies (Cedarleaf rams (initial BW = 83.0 ± 9.7 and 93.5 ± 9.14 kg, et  al., 1983; Johnson et  al., 1985). Avoidance to respectively) were used. For the duration of both bitter tasting plants is a mechanism sheep utilize trials, rams were housed indoors in individual pens, to limit toxin ingestion (Launchbaugh et  al., so feed and water intake could be monitored under 2001). Early research on the primary taste groups controlled conditions of 10 °C with a 12 h light:dark of sweet, sour, salty, and bitter in sheep suggested cycle. Additionally, feed and water were withheld that bitterness may be the most sensitive (Goatcher from rams from 1700 to 0700 h each day during the and Church, 1970a). Additional studies indicated trials. For each trial, rams were randomly allotted that sheep can taste and(or) sense bitterness when within breed to alternate pens throughout the barn. mimicked by addition of compounds, like quinine, Both trials were divided into two phases; an ac- when added to drinking water (Goatcher and climation phase, where rams were adjusted to the Church, 1970a; Favreau et  al., 2010), and lithium pens and daily feed and fluid delivery routines, and chloride, when added to forages (Launchbaugh and a testing period, where the phenylthiocarbamide Provenza, 1994). (PTC) treatments were delivered. Phenylthiocarbamide (PTC) is a compound, not found in nature, that mimics bitter tastes found Experimental Design in food (Blakeslee and Salmon, 1935; Barnicot et al., 1951; Lee and O’Mahony, 1998), and has been Both trials were conducted as a cross-over de- used in bitter taste research in humans (Blakeslee, sign consisting of five PTC treatments with indi- 1932; Fox, 1932; Harris and Kalmus, 1949) and vidual rams receiving a different PTC concentration mice (Lush, 1986; Nelson et al., 2003). In humans, each test day. In order for all rams to be tested in a PTC thresholds have been suggested to be heritable day, rams were randomized to five testing blocks Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/3/4/txz125/5581782 by Ed 'DeepDyve' Gillespie user on 18 October 2019 Phenylthiocarbamide avoidance in rams 3 Table 1. Sequence in which blocks of rams received each PTC solution concentration for both trials Test day Trial 1—PTC concentrations (mM) 0.20 0.56 1.57 4.39 12.29 1 Group 1 Group 2 Group 3 Group 4 Group 5 2 Group 2 Group 5 Group 4 Group 1 Group 3 3 Group 5 Group 3 Group 1 Group 2 Group 4 4 Group 3 Group 4 Group 2 Group 5 Group 1 5 Group 4 Group 1 Group 5 Group 3 Group 2 Trial 2—PTC concentrations (mM) 0.20 0.43 0.94 2.03 4.39 1 Group 1 Group 2 Group 3 Group 4 Group 5 2 Group 2 Group 5 Group 4 Group 1 Group 3 3 Group 5 Group 3 Group 1 Group 2 Group 4 4 Group 3 Group 4 Group 2 Group 5 Group 1 5 Group 4 Group 1 Group 5 Group 3 Group 2 Each group consisted of 3 rams with a total of 15 rams tested on each test day per trial. consisting of three rams each. Each block was ran- Table 2.  Alfalfa pellets and mineral supplement domly assigned a PTC testing sequence, which con- component analysis (DM basis) sisted of the order in which rams received their PTC a b Item Alfalfa pellets Mineral supplement treatments over the five test days (Table 1). Dry matter, % 100 100 Each trial consisted of a 5-d acclimation phase Crude protein, % 17.4 — followed by the PTC testing. During the acclima- Acid detergent fiber, % 36.8 — tion phase of both trials, rams received alfalfa pel- Total digestible nutrients 54.8 — lets (Table 2) at a rate of 1.9% of BW (as fed basis) Ca, % 1.79 0.85 at 0700  h. Thirty minutes after feeding, feed was P, % 0.22 0.002 removed, and refusals weighed. Immediately fol- K, % 2.09 0.03 lowing feed removal, two buckets filled with water Mg, % 0.29 0.06 (1.5 kg, trial 1; 3.0 kg, trial 2) were placed in each S, % 0.28 0.07 pen and rams were allowed access to the water for Na, % 0.16 95.0 Zn, mg/kg 22.6 1 1 h in trial 1 and 2 h in trial 2. Buckets were placed Fe, mg/kg 717 300 side-by-side in a holding rack and given a designa- Mn, mg/kg 50 5 tion of left and right side. At the end of the first Cu, mg/kg 7.8 3 water consumption period, buckets were removed Mo, mg/kg 2.17 — from each pen and water refusals weighed for each bucket and discarded. After removal of buckets, Component analysis of alfalfa pellets conducted by Ward Labora- tories (Kearney, NE). pens were cleaned, and rams were given their daily Mineral supplement formulated by Redmond Agriculture feed and water at approximately 0900 h for trial 1 (Redmond, UT). Product name “10 Fine Premium Mineral Salt.” and 1000 h for trial 2. In trial 1, rams received alfalfa pellets (Table 2) fed at a rate of 2.8% of BW (as fed basis), 45 g of a mineral mix (Table 2), and 5 kg of PTC concentration mimics the degree of bitterness water in a single bucket. At approximately 1230 h, in plants; therefore, PTC concentrations for trial 1 the water bucket was removed, refusals weighed, were chosen over a large range, then adjusted for and discarded. An additional 4  kg of water was trial 2 to better meet the objectives of the study. In offered, and at 1700 h, all feed and water were re- trial 1, some individuals consumed all fluid in either moved, refusals weighed, and discarded. Whereas bucket offered during the testing times. Therefore, in trial 2, rams received alfalfa pellets fed at a rate in order to limit thirst as a potential factor in con- of 2.8% of BW (as fed basis), 45 g of a mineral mix, sumption, the volume of water and PTC solution and two buckets with each containing 4 kg of water offered were increased while the time allotted for were offered, and at 1700 h, all feed and water were consumption was also increased for trial 2. removed, refusals weighed, and discarded. The test phase for both trials consisted of test The PTC was chosen as a bitter tasting agent days where PTC solutions and water (in separate to mimic the attributes of monoterpenoids, which buckets) were delivered after the morning feed- are often found in toxic shrubs. It is unknown what ing, and each test day was followed by a rest day Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/3/4/txz125/5581782 by Ed 'DeepDyve' Gillespie user on 18 October 2019 4 Henslee et al. where only water was delivered to minimize poten- were made using pair-wise contrasts (PDIFF). tial carry-over effects of PTC from the previous Significance was set at P ≤ 0.05. test day. Tap water (water) from the USSES well Due to the variation in avoidance to PTC ob- was used for this study. For test and rest days, the served within and across PTC concentrations for same procedures relative to timing of feed delivery, each trial (Table 3), individual rams were further number of buckets, and total amounts of fluid de- classified into consumer groups based upon total livered were followed as per the acclimation phase. PTC intake (g) over the five test days. Consumer On a test day, each ram block received one of the group differentiation was determined by 0.5 five concentrations of PTC solutions (trial 1: 0.20, standard deviation of the population mean to divide 0.56, 1.57, 4.39, or 12.29  mM delivered in a total rams into high (≥0.5 SD), medium (<0.5 to >−0.5 volume of 1.5 kg; trial 2: 0.20, 0.43, 0.94, 2.03, or SD), or low (≤−0.5 SD) PTC consumers (Table 4). 4.39 mM delivered in a total volume of 3.0 kg) in One objective of this study was to evaluate vari- one bucket, and water only (trial 1: 1.5 kg; trial 2: ation among individual rams. To test the variation 3.0  kg) in the other bucket. The location (left or observed, linear regression using PROC GLM for right) of the PTC solution bucket was alternated analysis by consumer group with the independent between test days. On the subsequent rest day, no variable being PTC concentration and dependent PTC solution was administered and was replaced variable included percent of PTC solution intake of with water. For both trials, PTC (Sigma P7629, test fluid intake. Orthogonal and paired contrasts Sigma–Aldrich, Saint Louis, MO) was dissolved in were used to test coincidence of regression lines absolute ethanol then diluted with water to the de- (slope and intercept analyzed together), as well as sired concentrations for delivery. slopes, and intercepts individually between PTC consumption groups. Statistical Analysis RESULTS AND DISCUSSION For all fluid intake variables analyzed within a trial, data were analyzed using PROC MIXED pro- We hypothesized sheep could detect PTC when cedures of SAS (Statistical Analysis System, SAS mixed in water and that sensitivity would be dif- Institute, Inc., Version 9.4, Cary, NC). The model ferent among individuals. Unlike in human stud- included treatment (PTC concentration), sequence ies (Fox, 1932; Blakeslee and Salmon, 1935), rams (order PTC concentrations were administered to are unable to verbally express if they can detect rams), and period (day that PTC was administered PTC. Although behavioral data were not quan- within the sequence) with a random statement that tified in this experiment, PTC concentrations included ram within sequence. Means are reported where the PTC solution was consumed less than as least squares means, and mean comparisons water negative behavioral reactions were observed Table 3. Descriptive statistics of variation observed across phenylthiocarbamide (PTC) concentration cat- egories within each trial where values are represented as percentage of PTC solution intake of total fluid offered and percentage of water intake of total fluid offered Trial 1 Coefficient of PTC concentration, mM Mean ± SD Minimum Maximum variation PTC Water PTC Water PTC Water PTC Water 0.20 54.5 ± 36.0 67.3 ± 35.9 0.5 1.0 95.7 100 66.1 53.3 0.56 39.0 ± 33.5 65.0 ± 35.8 0.4 1.0 93.1 100 86.0 55.0 1.57 30.9 ± 32.4 71.5 ± 32.5 0.5 8.2 94.7 100 105.0 45.4 4.39 7.2 ± 9.1 81.2 ± 26.9 0.3 0.6 32.2 100 126.7 33.1 12.29 3.6 ± 4.0 77.7 ± 26.0 0.7 25.9 14.3 100 112.1 33.5 Trial 2 0.20 41.9 ± 33.8 92.7 ± 10.1 0.3 69.1 97.6 100 80.8 10.9 0.43 29.9 ± 23.9 88.0 ± 22.0 0.3 28.6 90.9 100 79.7 25.0 0.94 40.5 ± 34.5 88.0 ± 18.3 0.5 39.6 95.8 100 85.1 20.8 2.03 33.1 ± 27.9 89.5 ± 17.3 0.3 53.0 89.8 100 84.3 19.3 4.39 11.5 ± 15.4 90.2 ± 16.7 0.5 49.2 57.1 100 133.3 18.5 All units are expressed as percentages. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/3/4/txz125/5581782 by Ed 'DeepDyve' Gillespie user on 18 October 2019 Phenylthiocarbamide avoidance in rams 5 during the study (e.g., smacking lips and shaking (P  <  0.001). The greatest decrease in percentage their head after tasting the PTC), particularly with of PTC solution intake was observed between 1.57 the highest PTC concentrations (data not shown). and 4.39 mM (58%) in trial 1. As PTC concentra- Furthermore, as PTC concentration increased, tion increased, PTC solution intake decreased, and mean intake of the PTC solutions decreased water intake increased (P < 0.001). (Tables 5 and 6). Individual reactions and intake of In trial 2, the greatest decrease in percentage PTC solution, taken altogether, suggest that rams of PTC solution intake was observed between 2.03 could detect PTC, and that animals varied in sensi- and 4.39 mM (72%). There was also a treatment ef- tivity to detection of PTC. fect (P < 0.01) on PTC solution intake but a slightly Consumption of PTC solution expressed as a different trend was observed than in trial 1.  The percentage of total morning fluid intake is depicted intake of the 0.20, 0.43, 0.94, and 2.03 concentra- in Tables 5 and 6 (trials 1 and 2, respectively). In tions were all similar (P > 0.05), but the intake of trial 1, as each solution increased in PTC con- the 4.39 mM concentration was different (P ≤ 0.05) centration the intake of PTC solution decreased than the rest. Similar to observations from trial 1, Table 4. Descriptive statistics of phenylthiocarbamide (PTC) consumption categories based on rankings of total PTC consumption by individual rams across the five test days within a trial Trial 1 Trial 2 Ram ID Consumption group Total PTC intake (g) Ram ID Consumption group Total PTC intake (g) T6581 High 1.056 T6886 High 2.103 S0791 High 0.876 T6342 High 1.389 S1497 High 0.769 S0801 High 1.318 S0583 Medium 0.408 T6884 High 1.236 T6406 Medium 0.384 T6093 High 1.163 S1500 Medium 0.305 T6516 Medium 0.932 T6885 Medium 0.294 T6313 Medium 0.781 T6578 Medium 0.281 S1499 Medium 0.737 T6502 Low 0.207 T6582 Medium 0.711 T6883 Low 0.205 T6299 Medium 0.697 T6297 Low 0.204 S1069 Low 0.452 S1501 Low 0.166 S0912 Low 0.327 S1125 Low 0.106 S1498 Low 0.177 S1124 Low 0.104 T6580 Low 0.091 T6401 Low 0.082 S1126 Low 0.035 Mean ± SD for all rams 0.363 ± 0.299 0.810 ± 0.567 Thresholds determined by mean ± (0.5 × SD). Table 5.  Mean fluid intakes of rams receiving either water or phenylthiocarbamide (PTC) during a test period for trial 1 PTC concentration, mM P values Pooled Variable 0.20 0.56 1.57 4.39 12.29 SE Treatment Sequence Period a a,b a,b b b Total test fluid intake as a percentage of total 60.9 52.0 51.2 44.2 40.7 6.3 0.02 0.74 0.33 offered (1.5 kg water and 1.5 kg PTC solution) a a,b b c c Water intake as a percentage of total test fluid in- 57.6 64.5 72.1 88.4 95.9 5.0 0.0001 0.39 0.11 take a a,b b c c PTC solution intake as a percentage of total test 42.4 35.5 27.9 11.6 4.1 5.0 0.0001 0.39 0.11 fluid intake Afternoon water intake as a percentage of total 92.5 91.4 91.6 89.5 92.9 3.2 0.61 0.68 0.03 offered (9 kg) a b b c b,c Total fluid intake as a percentage of total fluid 84.6 81.5 81.5 78.2 79.9 2.6 0.003 0.54 0.002 offered (12 kg) Treatment refers to PTC concentrations, sequence is the order PTC concentrations that were administered to rams, and period is the day PTC was administered within the sequence. a,b,c Means with different superscripts within a response and across PTC concentrations are different (P ≤ 0.05). Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/3/4/txz125/5581782 by Ed 'DeepDyve' Gillespie user on 18 October 2019 6 Henslee et al. Table 6.  Mean fluid intakes of rams receiving either water or phenylthiocarbamide (PTC) during a test period for trial 2 PTC concentration, mM P values Pooled Variable 0.20 0.43 0.94 2.03 4.39 SE Treatment Sequence Period a b a,b a,b c Total test fluid intake as a percentage of total 67.3 58.9 64.3 61.3 50.8 4.5 0.0002 0.86 0.02 offered (1.5 kg water and 1.5 kg PTC solution) a a a a b Water intake as a percentage of total test fluid in- 73.6 75.9 72.9 76.3 90.3 4.8 0.01 0.98 0.87 take a a a a b PTC solution intake as a percentage of total test 26.4 24.1 27.1 23.7 9.7 4.8 0.01 0.98 0.87 fluid intake Afternoon water intake as a percentage of total 86.2 91.7 94.1 95.6 95.9 2.7 0.06 0.07 0.01 offered (9 kg) Total fluid intake as a percentage of total fluid 78.1 77.7 81.3 80.9 76.6 2.5 0.25 0.46 0.005 offered (12 kg) Treatment refers to PTC concentrations, sequence is the order PTC concentrations were administered to rams, and period is the day PTC was administered within the sequence. a,b Means with different superscripts within a variable and across PTC concentrations are different (P ≤ 0.05). intake of the water increased as PTC increased in (1970a) administered increasing concentrations trial 2. The limited dose response in trial 2 may be of quinine (a bitter-mimicking agent) in drinking due to the smaller differences between PTC con- water to rams alongside a control of water and ob- centration levels for that trial and/or the increase served an inverse relationship between concentra- in total morning fluid offered. Trial 1 PTC concen- tion of quinine and percent of quinine solution of trations were chosen at approximately multiples of fluid intake. Goatcher and Church (1970b) further three; whereas in trial 2 PTC concentration were studied the sensitivity to quinine in a subsequent chosen at approximately multiples of two. Trial 1 study, and when analyzed on an individual basis. and trial 2 results indicated that the greatest de- Similar to this study, considerable variation from crease in PTC solution consumption occurred the mean was observed in percentage of solution in- when PTC concentrations increased from 1.57 to take of test fluid intake for each concentration. This 4.39  mM, which suggests a threshold within this large degree of difference in sensitivity among indi- range may have possible implications in determin- viduals has also been observed in human research ing bitter taste avoidance in sheep. and has led to the categorization of individuals into The lowest PTC concentration (0.20  mM) for tasters, and nontasters (Fox, 1932; Blakeslee and both trials had lower intake than the water. This Salmon, 1935). Research in humans has typically observation suggests PTC is detectable and avoid- placed participants into upper or lower thresholds ance begins for some rams below 0.20  mM. This to categorize tasters, nontasters, and super tasters, is supported by the observation that minimum which was originally suggested by Bartoshuk et al. consumption of the 0.20  mM PTC solution were (1994). Tasters are categorized as “tasters” if they 0.5% and 0.3% of total fluid intake for trials 1 and can detect PTC at a low concentration and as “non- 2, respectively (Table 3). It should also be noted, tasters” when detection is not until they consume however, that within the 0.20  mM PTC concen- a high concentration (Blakeslee and Salmon, 1935; tration that maximum PTC solution consumption Harris and Kalmus, 1949). The lack of standardiza- was >95% for trials 1 and 2 (Table 3). In trial 2, tion of testing sensitivity to PTC has produced in- the mean consumption of the 0.20 mM PTC con- consistent conclusions (Tepper, 2008). In this study, centration was approximately half of that observed some individuals (tasters) consumed less than 1% in trial 1. This difference could be attributed to the of the 0.20 mM PTC concentration, and other indi- amount of time allotted to consume test fluid. If viduals (nontasters) consumed >95% in both trials. a ram chose to avoid a particular PTC concentra- Including the observations made by Goatcher and tion, there would be more time in trial 2 to consume Church (1970a, 1970b) and those from this study, water postavoidance. sheep might fall into similar categories as humans. Nelson et  al. (2003) observed a similar in- While it is known that sheep will tolerate bit- verse relationship between PTC concentration and ter-tasting compounds (Provenza et  al., 1992; average intake when PTC solutions were admin- Launchbaugh et  al., 2001), there is no previous istered to mice. Similarly, Goatcher and Church literature indicating PTC tolerance thresholds in Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/3/4/txz125/5581782 by Ed 'DeepDyve' Gillespie user on 18 October 2019 Phenylthiocarbamide avoidance in rams 7 sheep. In human research, PTC categories have low consisted of seven, where in trial 2, all groups been suggested to be associated with bitterness in- consisted of five individuals. tensity perception (Blakeslee and Salmon, 1935; Similar to the sensitivity observed in this study, Bartoshuk et  al., 1994; Drewnowski and Rock, sensitivity to consuming bitter shrubs has also 1995). However, quinine sensitivity and PTC sen- been observed in grazing sheep. Snowder et  al. sitivity in humans are variable where some individ- (2001) determined percentage of sagebrush con- uals perceive quinine as being more bitter than PTC sumed in the diet of 549 ewes was 10.3–31.9% and some individuals perceive PTC as being more for September and 23.7–42.3% for October. The bitter than quinine (Blakeslee and Salmon, 1935; September and October measurements were highly Frank and Korchmar, 1985). The bitter tasting correlated (r  = 0.91), where the highest consumers compound PTC contains a thiocyanate moiety in September were also the highest consumers in (Bartoshuk et al., 1994), which is similar to isothio- October, similar to this study, where the individuals cyanates. Isothiocyanates are produced during the in the high consumer group consistently consumed breakdown of glucosinolates, commonly found in the most PTC solution. bitter tasting vegetables (Ettlinger and Lundeen, Variation was also observed in total daily fluid 1957). Quinine and PTC both elicit bitter tastes, but intake among the rams in this study. Based on this likely due to its’ chemical makeup, PTC sensitivity observation, to account for individual total fluid has been linked to glucosinolates preference (Duffy intake variation, we used the percent of PTC solu- and Bartoshuk, 2000). Goatcher and Church tion intake of total morning fluid intake (Figures (1970a) observed a similar inverse relationship be- 1 and 2, and Tables 5 and 6). Regression analyses tween increasing quinine solution concentrations were performed on each consumer group within and decrease in consumption as described in this each trial based on percentage of PTC solution in- study, but sensitivity to quinine or PTC may trans- take of total morning fluid intake (Figures 1 and late differently to foraging preferences in sheep. 2). The slopes of each consumer group within each For both trials, there were no sequence effects trial did not differ (P > 0.05), suggesting that the (P > 0.05) observed for percentage of PTC solu- rate of avoidance between consumer groups was tion consumed, indicating that the sequence in not different. However, most of the intercepts which rams received the PTC solutions did not af- differed across consumer groups (Figures 1 and fect fluid intakes on subsequent test days (Tables 2), which suggests that the point of avoidance as 4 and 5). There was also no sequence effect (P > PTC concentration increases is different between 0.05) on total fluid intake on rest days, which sug- groups. gested the effects of PTC dissipate rapidly (data In trial 1, within the medium and low groups, not shown). Relative to both PTC treatment and no individual consumed more than 5% of the sequence that rams received it on a test day, there highest PTC concentration (12.29  mM) offered. were no treatment or sequence effects (P > 0.05) Because the point at which the greatest avoidance on the amount of water intake during the rest days within the population was observed between the for the morning, afternoon, and total fluid intake. 1.57 and 4.39  mM PTC concentrations in trial 1, The average percentage of water intake on the rest the 12.29  mM concentration was eliminated for days during the morning, afternoon, and total for trial 2.  Furthermore, because the range of PTC the day were 56.8% ± 7.7%, 56.8% ± 3.5%, and concentrations for trial 2 was smaller than that 82.6% ± 3.0%, respectively for trial 1 and 76.6% ± of trial 1, the amount of PTC-solution and water 4.7%, 90.1% ± 3.0%, and 84.3% ± 2.3 %, respect- offered in the morning and the time allotted for in- ively for trial 2. take were increased. These changes made from trial A great deal of variation in PTC-solution intake 1 to trial 2 were to encourage those individuals that was observed between rams (Table 4). In trial 1, the were willing to consume greater concentrations of ram with the greatest intake of PTC consumed 9.7- PTC to differentiate themselves from the popula- fold more PTC than the ram with the lowest intake tion. Although PTC concentrations, total fluid (1.06 vs. 0.109 g, respectively). For trial 2, the mag- offered, and duration that the PTC solution was nitude of difference was much greater at 60-fold available to the rams varied between trials, a similar (2.10 vs. 0.0348 g PTC, respectively). Based on the individual variation in PTC solution intake was still variation between rams within each trial, rams were observed in trial 2 compared with trial 1 (Figures grouped according to total (g) PTC intake (Table 1 and 2). These results suggest that PTC intake is 4). In trial 1, the high intake group consisted of related to the individual ram’s preference for, or three individuals, medium consisted of five, and avoidance to, bitter taste and that PTC can be used Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/3/4/txz125/5581782 by Ed 'DeepDyve' Gillespie user on 18 October 2019 8 Henslee et al. Figure 1. Trial 1 linear regressions of phenylthiocarbamide (PTC) consumption by total PTC intake categories (high: □ − − −; medium: × ——; low: ○ ∙ ∙ ∙ ∙) for Rambouillet and Targhee rams administered five PTC concentrations (0.20, 0.56, 1.57, 4.39, and 12.29 mM) suspended in 1.5 kg of water. Paired contrast made between high vs. low, medium vs. low, and low vs. high. Orthogonal contrast made between high/medium vs. low and high vs. medium/low. Figure 2. Trial 2 linear regressions of phenylthiocarbamide (PTC) consumption by total PTC intake categories (high: □ − − −; medium: × ——; low: ○ ∙ ∙ ∙ ∙) for Rambouillet and Targhee rams administered five PTC concentrations (0.20, 0.43, 0.94, 2.03, and 4.39 mM) suspended in 3.0 kg of water. Paired contrast made between high vs. low, medium vs. low, and low vs. high. Orthogonal contrast made between high/medium vs. low and high vs. medium/low. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/3/4/txz125/5581782 by Ed 'DeepDyve' Gillespie user on 18 October 2019 Phenylthiocarbamide avoidance in rams 9 as a bitter-mimicking agent to determine sensitivity (Sclafani, 1991; Provenza et al., 1992; Launchbaugh to bitterness among individuals. et  al., 2001). Differences in memory of a forage is To date, this is the first study to use PTC to test likely linked to the individual’s physiologic ability for sensitivity of bitterness among sheep. Sheep have to suppress the toxic effects (Provenza et al., 1992). displayed the ability to identify the presence of terpe- Because terpenoids contain bitter-tasting com- nes when fed in a mixed ration (Villalba et al., 2006; pounds, variation in bitter preference between indi- Mote et  al., 2007), which suggests that part of diet viduals may not only translate to forage selection but selection is sensory and is related to taste and(or) may also be correlated with the individual’s ability aroma. Dziba and Provenza (2008) reported that in to suppress toxins. Toxic shrub intake is likely driven lambs offered varying concentrations of monoter- by several phenotypic (Mennella et al., 2005; Dziba penoids (camphor, p-cymeme,1–8 cineole, methacr- et al., 2007; Ginane et al., 2011) and genotypic fac- olein; commonly found in Big Mountain sagebrush) tors (Chandrashekar et al., 2000; Bufe et al., 2005). mixed into their diets, intake rates of the mixed diet in Results from this study indicate that there are relation to monoterpene concentration varied. There sensitivity differences between individual’s preference was no difference in the percentage of time spent eat- for consuming bitter tasting compounds in sheep. ing among the groups (high concentration  =  4.65% The variation in bitterness intake may translate to terpene, medium concentration  =  3.10%, and low foraging preferences while grazing, where rams with concentration = 1.55%), but the medium group con- greater tolerance for bitter taste may consume plants sumed less than the low group and the high group with higher concentrations of bitter tasting com- consumed less than the medium group. Furthermore, pounds, such as monoterpenoids. Similarly, humans Dziba and Provenza (2008) suggested that lambs that are categorized as nontasters consume more regulate feed intake of bitter vegetation to prevent anti-oxidant rich vegetables with bitter attributes consuming a toxic dose of terpenes. Although total than tasters (Garcia-Bailo et al., 2009). amount of forages consumed differed between the Utilizing sheep as a grazing tool to reduce sage- medium and high group, both groups stopped con- brush canopy has been suggested to entail long-term suming feed when they reached approximately 28  g and high-intensity grazing applications (Seefeldt, of monoterpenoids per day (Dziba and Provenza, 2005); however, sheep grazing may be a good tool 2008). Launchbaugh et  al. (1993) observed similar for suppressing sagebrush canopy growth and de- behavior when lithium chloride was mixed in diets crease shrub encroachment on grasslands. Moffet fed to lambs. Despite the concentration at which et  al. (2015) suggested that during a rangeland lithium chloride was fed, lambs regulated their life cycle in a mountain big sagebrush ecosystem, total intake to not exceed concentrations of 62.7  ± the greatest forage productivity and optimal wild- 4.5  mg/kg lithium chloride per day. Regulating in- life habitat conditions occur 5–15  years postfire. take of bitter-tasting compounds is likely a devel- Furthermore, productivity of rangeland decreases oped mechanism that sheep use to avoid forages that as sagebrush canopies become overgrown. Johnson have negative postingestion qualities (Provenza and et  al. (1996) suggested that the greatest ecological Balph, 1987). While postingestion feedback is one diversity in mountain big sagebrush ecosystems oc- mechanism used by ruminants in forage selection, it curs when the sagebrush canopy makes up approxi- is likely not the only deciding factor. mately 15% of total plant composition, and the Launchbaugh (2001) suggested that foraging greatest herbaceous production occurs when the behaviors can be learned from mimicking maternal sagebrush canopy makes up 11–17% of total plant and herd behavior, and taste memory from suck- composition. Because diet selection is moderately ling. Nolte and Provenza (1992) observed that feed- heritable in sheep (h  = 0.28) (Snowder et al., 2001), ing onion-flavored milk to orphan lambs resulted selection for sheep that have a higher tolerance for in a preference for onion-flavored feeds later in life. bitter tasting compounds may translate to sage- Some literature suggests that bitterness is likely not brush canopy growth suppression on rangeland, the apparent causative factor when consuming toxic and therefore, extend the optimal ecological pro- forages, but rather postingestive feedback mech- ductivity-time period beyond 5–15  years postfire anisms (Provenza et  al., 1992; Launchbaugh et  al., (Moffet et al., 2015). 2001). Future selection or determent of a forage is associated with the memory of that taste and the ACKNOWLEDGMENTS digestive feedback; however, memory and toler- ance vary between individuals and each individual The authors would like to acknowledge perceives cost/benefit from a forage differently the support for this research provided by the Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/3/4/txz125/5581782 by Ed 'DeepDyve' Gillespie user on 18 October 2019 10 Henslee et al. Favreau,  A., R.  Baumont, A.  J.  Duncan, and C.  Ginane. USDA-ARS-Sheep Experiment Station employees, 2010. Do sheep use umami and bitter tastes as cues of Mark Williams, Taylor Hudson, and Ashleigh post-ingestive consequences when selecting their diet? Redman. The authors would also like to thank App. Anim. Behav. Sci. 125(3–4):115–123. doi:10.1016/j. 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Behav. 31:165– digestibility, intake, and preference in sheep. J. Anim. Sci. 172. doi:10.3758/bf03195979 84:2463–2473. doi:10.2527/jas.2005-716 Provenza,  F.  D., and D.  F.  Balph. 1987. Diet learning by do- Wambolt,  C.  L., and G.  F.  Payne. 1986. An 18-year com- mestic ruminants—theory, evidence and practical im- parison of control methods for Wyoming big sagebrush in plications. App. Anim. Behav. Sci. 18(3–4):211–232. Southwestern Montana. J. Range Management 39(4):314– doi:10.1016/0168-1591(87)90218-8 319. doi:10.2307/3899770 Translate basic science to industry innovation http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Translational Animal Science Oxford University Press

Avoidance of phenylthiocarbamide in mature Targhee and Rambouillet rams

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© The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science.
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Downloaded from https://academic.oup.com/tas/article-abstract/3/4/txz125/5581782 by Ed 'DeepDyve' Gillespie user on 18 October 2019 † ‡, ,1 Dillan Henslee,* Joel Yelich, J. Bret Taylor, and Melinda Ellison* *Department of Animal and Veterinary Science, University of Idaho, Moscow, ID 83844; Department of Animal and Veterinary Science, Idaho Agricultural Experiment Station, Nancy M. Cummings Research, Extension and Education Center, University of Idaho, Carmen, ID 83462; and U.S. Sheep Experiment Station, Agricultural Research Service, USDA, Dubois, ID 83423 ABSTRACT:  Shrub encroachment on grass- 2  h in trial 2.  Each test day was followed by a lands is a worldwide issue and sheep are a po- rest day where PTC solution was replaced with tential tool for mitigating shrub encroachment. water to limit potential carry over effects into Many shrubs, however, contain bitter-tasting the next test day. Consumption of PTC solution compounds that may deter grazers. Cattle and for each PTC concentration was expressed as sheep commonly graze rangelands, but of the the percentage of PTC solution intake of total two, sheep have a greater tolerance for bitter morning fluid intake. There was no effect (P > compounds and would be expected to consume 0.74) of sequence that rams received PTC solu- more bitter-tasting vegetation. We hypothesized tions on PTC consumption during either trial. that sheep could detect (i.e., taste) bitter-tasting As PTC concentration increased, percentage compounds and the sensitivity to these com- of PTC solution intake decreased (P ≤ 0.01) pounds would vary from animal to animal. The for both trials. The greatest decrease in per- objective of this study was to determine whether centage of PTC solution intake occurred be- sheep could detect the bitter-tasting compound tween 1.57 and 4.39  mM (58%) for trial 1 and phenylthiocarbamide (PTC), and if so, what 2.03 and 4.39  mM (72%) for trial 2.  In trial 2, PTC concentration would elicit an avoidance the least percentage of PTC solution intake was response. Using a crossover study design, ma- the 4.39  mM PTC concentration, which was ture Rambouillet and Targhee rams (n  =  30) different (P ≤ 0.05) from lesser PTC concen- were subjected in randomized order to various trations. All other PTC concentrations did not PTC concentrations mixed in the drinking water differ (P > 0.05) from each other in percentage (PTC solution). In trials 1 and 2 (n  =  15/trial), intake. This research suggests rams could taste 0.20, 0.56, 1.57, 4.39, and 12.29  mM and 0.20, the PTC, and the concentration at which PTC 0.43, 0.94, 2.03, and 4.39  mM of PTC were solution was avoided varied across rams. It may tested, respectively. On test days, PTC solution be possible to select sheep, based on demon- (trial 1: 1.5 kg; trial 2: 3.0 kg) and water (same strated avoidance of PTC, for targeted grazing amounts) were offered for ad libitum intake in applications to manipulate vegetation toward a side-by-side presentation for 1 h in trial 1 and range management goals. Key words: bitterness avoidance, phenylthiocarbamide, sagebrush, sheep © The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Transl. Anim. Sci. 2019.XX:0-0 doi: 10.1093/tas/txz125 Corresponding author: ellison@uidaho.edu Received April 18, 2019. Accepted August 7, 2019. 1 Downloaded from https://academic.oup.com/tas/article-abstract/3/4/txz125/5581782 by Ed 'DeepDyve' Gillespie user on 18 October 2019 2 Henslee et al. INTRODUCTION (h   =  0.55) (Morton et  al., 1981). It has also been suggested that PTC avoidance is influenced by post- Overgrowth of mountain big sagebrush digestive factors (Nelson et al., 2003), similar to the (Artemisia tridentate Nutt. ssp. vaseyana) can lead preferences of sheep grazing bitter/toxic forages to a reduction in rangeland plant diversity, carrying (Launchbaugh et al., 2001). capacity, and wildlife abundance (Johnson et  al., This study focused on bitter taste avoidance 1996; Launchbaugh, 2003). Sagebrush can be con- (Parker, 2003) by the addition of PTC in water. We trolled or eliminated by plowing, burning, and hypothesized that sheep could detect bitter-tasting spraying (Wambolt and Payne, 1986), but these compounds and the sensitivity would vary from methods can be expensive and have potential un- animal to animal. The objective of this study was desirable effects on rangelands. One method of to determine whether sheep could detect the bitter control that has received minimal attention, and tasting compound, phenylthiocarbamide, and if so, may be more sustainable, is reduction of sagebrush what PTC concentration would elicit an avoidance with grazing sheep. Using fecal analysis over sev- response. eral experiments, Snowder et  al. (2001) indicated that the dietary preference for sagebrush in sheep MATERIALS AND METHODS has a heritability of 0.28, suggesting that selection against bitterness avoidance in sheep breeding pro- Animals grams may be feasible. Furthermore, Ferreira et al. (2013) identified a set of novel genes for bitter taste All animal procedures were approved by an receptors in sheep, suggesting that sheep may be Institutional Animal Care and Use Committee genetically predisposed to select or avoid plants (USDA, ARS, Dubois, ID) in accordance with the with bitter or noxious tastes. USDA, APHIS Animal Welfare Regulations (2013; Sheep are adaptive selective grazers 9 C.F.R. § 2.30-2.38 2013) and the Guide for the Care (Launchbaugh et  al., 2001) with varying dietary and Use of Agricultural Animals in Research and preference for consuming sagebrush (Bork et  al., Teaching (FASS, 2010). Two trials were conducted 1998; Snowder et al., 2001; Seefeldt, 2005). Several at the USDA, ARS U.S. Sheep Experiment Station factors can be attributed to an individual’s diet pref- (USSES) located near Dubois, Idaho in the spring erence/selection including learned behaviors, taste of 2018. In trial 1, yearling Rambouillet (n = 7) and preference, postdigestive feedback, and their ability Targhee (n = 8) rams (initial BW = 76.6 ± 5.7 and to detoxify secondary metabolites. Many toxic for- 83.7 ± 9.1 kg, respectively) were used; while in trial ages have a bitter taste, but the toxicity and the cor- 2, yearling Rambouillet (n = 6) and Targhee (n = 9) relation of bitter taste to toxicity varies (Cedarleaf rams (initial BW = 83.0 ± 9.7 and 93.5 ± 9.14 kg, et  al., 1983; Johnson et  al., 1985). Avoidance to respectively) were used. For the duration of both bitter tasting plants is a mechanism sheep utilize trials, rams were housed indoors in individual pens, to limit toxin ingestion (Launchbaugh et  al., so feed and water intake could be monitored under 2001). Early research on the primary taste groups controlled conditions of 10 °C with a 12 h light:dark of sweet, sour, salty, and bitter in sheep suggested cycle. Additionally, feed and water were withheld that bitterness may be the most sensitive (Goatcher from rams from 1700 to 0700 h each day during the and Church, 1970a). Additional studies indicated trials. For each trial, rams were randomly allotted that sheep can taste and(or) sense bitterness when within breed to alternate pens throughout the barn. mimicked by addition of compounds, like quinine, Both trials were divided into two phases; an ac- when added to drinking water (Goatcher and climation phase, where rams were adjusted to the Church, 1970a; Favreau et  al., 2010), and lithium pens and daily feed and fluid delivery routines, and chloride, when added to forages (Launchbaugh and a testing period, where the phenylthiocarbamide Provenza, 1994). (PTC) treatments were delivered. Phenylthiocarbamide (PTC) is a compound, not found in nature, that mimics bitter tastes found Experimental Design in food (Blakeslee and Salmon, 1935; Barnicot et al., 1951; Lee and O’Mahony, 1998), and has been Both trials were conducted as a cross-over de- used in bitter taste research in humans (Blakeslee, sign consisting of five PTC treatments with indi- 1932; Fox, 1932; Harris and Kalmus, 1949) and vidual rams receiving a different PTC concentration mice (Lush, 1986; Nelson et al., 2003). In humans, each test day. In order for all rams to be tested in a PTC thresholds have been suggested to be heritable day, rams were randomized to five testing blocks Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/3/4/txz125/5581782 by Ed 'DeepDyve' Gillespie user on 18 October 2019 Phenylthiocarbamide avoidance in rams 3 Table 1. Sequence in which blocks of rams received each PTC solution concentration for both trials Test day Trial 1—PTC concentrations (mM) 0.20 0.56 1.57 4.39 12.29 1 Group 1 Group 2 Group 3 Group 4 Group 5 2 Group 2 Group 5 Group 4 Group 1 Group 3 3 Group 5 Group 3 Group 1 Group 2 Group 4 4 Group 3 Group 4 Group 2 Group 5 Group 1 5 Group 4 Group 1 Group 5 Group 3 Group 2 Trial 2—PTC concentrations (mM) 0.20 0.43 0.94 2.03 4.39 1 Group 1 Group 2 Group 3 Group 4 Group 5 2 Group 2 Group 5 Group 4 Group 1 Group 3 3 Group 5 Group 3 Group 1 Group 2 Group 4 4 Group 3 Group 4 Group 2 Group 5 Group 1 5 Group 4 Group 1 Group 5 Group 3 Group 2 Each group consisted of 3 rams with a total of 15 rams tested on each test day per trial. consisting of three rams each. Each block was ran- Table 2.  Alfalfa pellets and mineral supplement domly assigned a PTC testing sequence, which con- component analysis (DM basis) sisted of the order in which rams received their PTC a b Item Alfalfa pellets Mineral supplement treatments over the five test days (Table 1). Dry matter, % 100 100 Each trial consisted of a 5-d acclimation phase Crude protein, % 17.4 — followed by the PTC testing. During the acclima- Acid detergent fiber, % 36.8 — tion phase of both trials, rams received alfalfa pel- Total digestible nutrients 54.8 — lets (Table 2) at a rate of 1.9% of BW (as fed basis) Ca, % 1.79 0.85 at 0700  h. Thirty minutes after feeding, feed was P, % 0.22 0.002 removed, and refusals weighed. Immediately fol- K, % 2.09 0.03 lowing feed removal, two buckets filled with water Mg, % 0.29 0.06 (1.5 kg, trial 1; 3.0 kg, trial 2) were placed in each S, % 0.28 0.07 pen and rams were allowed access to the water for Na, % 0.16 95.0 Zn, mg/kg 22.6 1 1 h in trial 1 and 2 h in trial 2. Buckets were placed Fe, mg/kg 717 300 side-by-side in a holding rack and given a designa- Mn, mg/kg 50 5 tion of left and right side. At the end of the first Cu, mg/kg 7.8 3 water consumption period, buckets were removed Mo, mg/kg 2.17 — from each pen and water refusals weighed for each bucket and discarded. After removal of buckets, Component analysis of alfalfa pellets conducted by Ward Labora- tories (Kearney, NE). pens were cleaned, and rams were given their daily Mineral supplement formulated by Redmond Agriculture feed and water at approximately 0900 h for trial 1 (Redmond, UT). Product name “10 Fine Premium Mineral Salt.” and 1000 h for trial 2. In trial 1, rams received alfalfa pellets (Table 2) fed at a rate of 2.8% of BW (as fed basis), 45 g of a mineral mix (Table 2), and 5 kg of PTC concentration mimics the degree of bitterness water in a single bucket. At approximately 1230 h, in plants; therefore, PTC concentrations for trial 1 the water bucket was removed, refusals weighed, were chosen over a large range, then adjusted for and discarded. An additional 4  kg of water was trial 2 to better meet the objectives of the study. In offered, and at 1700 h, all feed and water were re- trial 1, some individuals consumed all fluid in either moved, refusals weighed, and discarded. Whereas bucket offered during the testing times. Therefore, in trial 2, rams received alfalfa pellets fed at a rate in order to limit thirst as a potential factor in con- of 2.8% of BW (as fed basis), 45 g of a mineral mix, sumption, the volume of water and PTC solution and two buckets with each containing 4 kg of water offered were increased while the time allotted for were offered, and at 1700 h, all feed and water were consumption was also increased for trial 2. removed, refusals weighed, and discarded. The test phase for both trials consisted of test The PTC was chosen as a bitter tasting agent days where PTC solutions and water (in separate to mimic the attributes of monoterpenoids, which buckets) were delivered after the morning feed- are often found in toxic shrubs. It is unknown what ing, and each test day was followed by a rest day Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/3/4/txz125/5581782 by Ed 'DeepDyve' Gillespie user on 18 October 2019 4 Henslee et al. where only water was delivered to minimize poten- were made using pair-wise contrasts (PDIFF). tial carry-over effects of PTC from the previous Significance was set at P ≤ 0.05. test day. Tap water (water) from the USSES well Due to the variation in avoidance to PTC ob- was used for this study. For test and rest days, the served within and across PTC concentrations for same procedures relative to timing of feed delivery, each trial (Table 3), individual rams were further number of buckets, and total amounts of fluid de- classified into consumer groups based upon total livered were followed as per the acclimation phase. PTC intake (g) over the five test days. Consumer On a test day, each ram block received one of the group differentiation was determined by 0.5 five concentrations of PTC solutions (trial 1: 0.20, standard deviation of the population mean to divide 0.56, 1.57, 4.39, or 12.29  mM delivered in a total rams into high (≥0.5 SD), medium (<0.5 to >−0.5 volume of 1.5 kg; trial 2: 0.20, 0.43, 0.94, 2.03, or SD), or low (≤−0.5 SD) PTC consumers (Table 4). 4.39 mM delivered in a total volume of 3.0 kg) in One objective of this study was to evaluate vari- one bucket, and water only (trial 1: 1.5 kg; trial 2: ation among individual rams. To test the variation 3.0  kg) in the other bucket. The location (left or observed, linear regression using PROC GLM for right) of the PTC solution bucket was alternated analysis by consumer group with the independent between test days. On the subsequent rest day, no variable being PTC concentration and dependent PTC solution was administered and was replaced variable included percent of PTC solution intake of with water. For both trials, PTC (Sigma P7629, test fluid intake. Orthogonal and paired contrasts Sigma–Aldrich, Saint Louis, MO) was dissolved in were used to test coincidence of regression lines absolute ethanol then diluted with water to the de- (slope and intercept analyzed together), as well as sired concentrations for delivery. slopes, and intercepts individually between PTC consumption groups. Statistical Analysis RESULTS AND DISCUSSION For all fluid intake variables analyzed within a trial, data were analyzed using PROC MIXED pro- We hypothesized sheep could detect PTC when cedures of SAS (Statistical Analysis System, SAS mixed in water and that sensitivity would be dif- Institute, Inc., Version 9.4, Cary, NC). The model ferent among individuals. Unlike in human stud- included treatment (PTC concentration), sequence ies (Fox, 1932; Blakeslee and Salmon, 1935), rams (order PTC concentrations were administered to are unable to verbally express if they can detect rams), and period (day that PTC was administered PTC. Although behavioral data were not quan- within the sequence) with a random statement that tified in this experiment, PTC concentrations included ram within sequence. Means are reported where the PTC solution was consumed less than as least squares means, and mean comparisons water negative behavioral reactions were observed Table 3. Descriptive statistics of variation observed across phenylthiocarbamide (PTC) concentration cat- egories within each trial where values are represented as percentage of PTC solution intake of total fluid offered and percentage of water intake of total fluid offered Trial 1 Coefficient of PTC concentration, mM Mean ± SD Minimum Maximum variation PTC Water PTC Water PTC Water PTC Water 0.20 54.5 ± 36.0 67.3 ± 35.9 0.5 1.0 95.7 100 66.1 53.3 0.56 39.0 ± 33.5 65.0 ± 35.8 0.4 1.0 93.1 100 86.0 55.0 1.57 30.9 ± 32.4 71.5 ± 32.5 0.5 8.2 94.7 100 105.0 45.4 4.39 7.2 ± 9.1 81.2 ± 26.9 0.3 0.6 32.2 100 126.7 33.1 12.29 3.6 ± 4.0 77.7 ± 26.0 0.7 25.9 14.3 100 112.1 33.5 Trial 2 0.20 41.9 ± 33.8 92.7 ± 10.1 0.3 69.1 97.6 100 80.8 10.9 0.43 29.9 ± 23.9 88.0 ± 22.0 0.3 28.6 90.9 100 79.7 25.0 0.94 40.5 ± 34.5 88.0 ± 18.3 0.5 39.6 95.8 100 85.1 20.8 2.03 33.1 ± 27.9 89.5 ± 17.3 0.3 53.0 89.8 100 84.3 19.3 4.39 11.5 ± 15.4 90.2 ± 16.7 0.5 49.2 57.1 100 133.3 18.5 All units are expressed as percentages. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/3/4/txz125/5581782 by Ed 'DeepDyve' Gillespie user on 18 October 2019 Phenylthiocarbamide avoidance in rams 5 during the study (e.g., smacking lips and shaking (P  <  0.001). The greatest decrease in percentage their head after tasting the PTC), particularly with of PTC solution intake was observed between 1.57 the highest PTC concentrations (data not shown). and 4.39 mM (58%) in trial 1. As PTC concentra- Furthermore, as PTC concentration increased, tion increased, PTC solution intake decreased, and mean intake of the PTC solutions decreased water intake increased (P < 0.001). (Tables 5 and 6). Individual reactions and intake of In trial 2, the greatest decrease in percentage PTC solution, taken altogether, suggest that rams of PTC solution intake was observed between 2.03 could detect PTC, and that animals varied in sensi- and 4.39 mM (72%). There was also a treatment ef- tivity to detection of PTC. fect (P < 0.01) on PTC solution intake but a slightly Consumption of PTC solution expressed as a different trend was observed than in trial 1.  The percentage of total morning fluid intake is depicted intake of the 0.20, 0.43, 0.94, and 2.03 concentra- in Tables 5 and 6 (trials 1 and 2, respectively). In tions were all similar (P > 0.05), but the intake of trial 1, as each solution increased in PTC con- the 4.39 mM concentration was different (P ≤ 0.05) centration the intake of PTC solution decreased than the rest. Similar to observations from trial 1, Table 4. Descriptive statistics of phenylthiocarbamide (PTC) consumption categories based on rankings of total PTC consumption by individual rams across the five test days within a trial Trial 1 Trial 2 Ram ID Consumption group Total PTC intake (g) Ram ID Consumption group Total PTC intake (g) T6581 High 1.056 T6886 High 2.103 S0791 High 0.876 T6342 High 1.389 S1497 High 0.769 S0801 High 1.318 S0583 Medium 0.408 T6884 High 1.236 T6406 Medium 0.384 T6093 High 1.163 S1500 Medium 0.305 T6516 Medium 0.932 T6885 Medium 0.294 T6313 Medium 0.781 T6578 Medium 0.281 S1499 Medium 0.737 T6502 Low 0.207 T6582 Medium 0.711 T6883 Low 0.205 T6299 Medium 0.697 T6297 Low 0.204 S1069 Low 0.452 S1501 Low 0.166 S0912 Low 0.327 S1125 Low 0.106 S1498 Low 0.177 S1124 Low 0.104 T6580 Low 0.091 T6401 Low 0.082 S1126 Low 0.035 Mean ± SD for all rams 0.363 ± 0.299 0.810 ± 0.567 Thresholds determined by mean ± (0.5 × SD). Table 5.  Mean fluid intakes of rams receiving either water or phenylthiocarbamide (PTC) during a test period for trial 1 PTC concentration, mM P values Pooled Variable 0.20 0.56 1.57 4.39 12.29 SE Treatment Sequence Period a a,b a,b b b Total test fluid intake as a percentage of total 60.9 52.0 51.2 44.2 40.7 6.3 0.02 0.74 0.33 offered (1.5 kg water and 1.5 kg PTC solution) a a,b b c c Water intake as a percentage of total test fluid in- 57.6 64.5 72.1 88.4 95.9 5.0 0.0001 0.39 0.11 take a a,b b c c PTC solution intake as a percentage of total test 42.4 35.5 27.9 11.6 4.1 5.0 0.0001 0.39 0.11 fluid intake Afternoon water intake as a percentage of total 92.5 91.4 91.6 89.5 92.9 3.2 0.61 0.68 0.03 offered (9 kg) a b b c b,c Total fluid intake as a percentage of total fluid 84.6 81.5 81.5 78.2 79.9 2.6 0.003 0.54 0.002 offered (12 kg) Treatment refers to PTC concentrations, sequence is the order PTC concentrations that were administered to rams, and period is the day PTC was administered within the sequence. a,b,c Means with different superscripts within a response and across PTC concentrations are different (P ≤ 0.05). Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/3/4/txz125/5581782 by Ed 'DeepDyve' Gillespie user on 18 October 2019 6 Henslee et al. Table 6.  Mean fluid intakes of rams receiving either water or phenylthiocarbamide (PTC) during a test period for trial 2 PTC concentration, mM P values Pooled Variable 0.20 0.43 0.94 2.03 4.39 SE Treatment Sequence Period a b a,b a,b c Total test fluid intake as a percentage of total 67.3 58.9 64.3 61.3 50.8 4.5 0.0002 0.86 0.02 offered (1.5 kg water and 1.5 kg PTC solution) a a a a b Water intake as a percentage of total test fluid in- 73.6 75.9 72.9 76.3 90.3 4.8 0.01 0.98 0.87 take a a a a b PTC solution intake as a percentage of total test 26.4 24.1 27.1 23.7 9.7 4.8 0.01 0.98 0.87 fluid intake Afternoon water intake as a percentage of total 86.2 91.7 94.1 95.6 95.9 2.7 0.06 0.07 0.01 offered (9 kg) Total fluid intake as a percentage of total fluid 78.1 77.7 81.3 80.9 76.6 2.5 0.25 0.46 0.005 offered (12 kg) Treatment refers to PTC concentrations, sequence is the order PTC concentrations were administered to rams, and period is the day PTC was administered within the sequence. a,b Means with different superscripts within a variable and across PTC concentrations are different (P ≤ 0.05). intake of the water increased as PTC increased in (1970a) administered increasing concentrations trial 2. The limited dose response in trial 2 may be of quinine (a bitter-mimicking agent) in drinking due to the smaller differences between PTC con- water to rams alongside a control of water and ob- centration levels for that trial and/or the increase served an inverse relationship between concentra- in total morning fluid offered. Trial 1 PTC concen- tion of quinine and percent of quinine solution of trations were chosen at approximately multiples of fluid intake. Goatcher and Church (1970b) further three; whereas in trial 2 PTC concentration were studied the sensitivity to quinine in a subsequent chosen at approximately multiples of two. Trial 1 study, and when analyzed on an individual basis. and trial 2 results indicated that the greatest de- Similar to this study, considerable variation from crease in PTC solution consumption occurred the mean was observed in percentage of solution in- when PTC concentrations increased from 1.57 to take of test fluid intake for each concentration. This 4.39  mM, which suggests a threshold within this large degree of difference in sensitivity among indi- range may have possible implications in determin- viduals has also been observed in human research ing bitter taste avoidance in sheep. and has led to the categorization of individuals into The lowest PTC concentration (0.20  mM) for tasters, and nontasters (Fox, 1932; Blakeslee and both trials had lower intake than the water. This Salmon, 1935). Research in humans has typically observation suggests PTC is detectable and avoid- placed participants into upper or lower thresholds ance begins for some rams below 0.20  mM. This to categorize tasters, nontasters, and super tasters, is supported by the observation that minimum which was originally suggested by Bartoshuk et al. consumption of the 0.20  mM PTC solution were (1994). Tasters are categorized as “tasters” if they 0.5% and 0.3% of total fluid intake for trials 1 and can detect PTC at a low concentration and as “non- 2, respectively (Table 3). It should also be noted, tasters” when detection is not until they consume however, that within the 0.20  mM PTC concen- a high concentration (Blakeslee and Salmon, 1935; tration that maximum PTC solution consumption Harris and Kalmus, 1949). The lack of standardiza- was >95% for trials 1 and 2 (Table 3). In trial 2, tion of testing sensitivity to PTC has produced in- the mean consumption of the 0.20 mM PTC con- consistent conclusions (Tepper, 2008). In this study, centration was approximately half of that observed some individuals (tasters) consumed less than 1% in trial 1. This difference could be attributed to the of the 0.20 mM PTC concentration, and other indi- amount of time allotted to consume test fluid. If viduals (nontasters) consumed >95% in both trials. a ram chose to avoid a particular PTC concentra- Including the observations made by Goatcher and tion, there would be more time in trial 2 to consume Church (1970a, 1970b) and those from this study, water postavoidance. sheep might fall into similar categories as humans. Nelson et  al. (2003) observed a similar in- While it is known that sheep will tolerate bit- verse relationship between PTC concentration and ter-tasting compounds (Provenza et  al., 1992; average intake when PTC solutions were admin- Launchbaugh et  al., 2001), there is no previous istered to mice. Similarly, Goatcher and Church literature indicating PTC tolerance thresholds in Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/3/4/txz125/5581782 by Ed 'DeepDyve' Gillespie user on 18 October 2019 Phenylthiocarbamide avoidance in rams 7 sheep. In human research, PTC categories have low consisted of seven, where in trial 2, all groups been suggested to be associated with bitterness in- consisted of five individuals. tensity perception (Blakeslee and Salmon, 1935; Similar to the sensitivity observed in this study, Bartoshuk et  al., 1994; Drewnowski and Rock, sensitivity to consuming bitter shrubs has also 1995). However, quinine sensitivity and PTC sen- been observed in grazing sheep. Snowder et  al. sitivity in humans are variable where some individ- (2001) determined percentage of sagebrush con- uals perceive quinine as being more bitter than PTC sumed in the diet of 549 ewes was 10.3–31.9% and some individuals perceive PTC as being more for September and 23.7–42.3% for October. The bitter than quinine (Blakeslee and Salmon, 1935; September and October measurements were highly Frank and Korchmar, 1985). The bitter tasting correlated (r  = 0.91), where the highest consumers compound PTC contains a thiocyanate moiety in September were also the highest consumers in (Bartoshuk et al., 1994), which is similar to isothio- October, similar to this study, where the individuals cyanates. Isothiocyanates are produced during the in the high consumer group consistently consumed breakdown of glucosinolates, commonly found in the most PTC solution. bitter tasting vegetables (Ettlinger and Lundeen, Variation was also observed in total daily fluid 1957). Quinine and PTC both elicit bitter tastes, but intake among the rams in this study. Based on this likely due to its’ chemical makeup, PTC sensitivity observation, to account for individual total fluid has been linked to glucosinolates preference (Duffy intake variation, we used the percent of PTC solu- and Bartoshuk, 2000). Goatcher and Church tion intake of total morning fluid intake (Figures (1970a) observed a similar inverse relationship be- 1 and 2, and Tables 5 and 6). Regression analyses tween increasing quinine solution concentrations were performed on each consumer group within and decrease in consumption as described in this each trial based on percentage of PTC solution in- study, but sensitivity to quinine or PTC may trans- take of total morning fluid intake (Figures 1 and late differently to foraging preferences in sheep. 2). The slopes of each consumer group within each For both trials, there were no sequence effects trial did not differ (P > 0.05), suggesting that the (P > 0.05) observed for percentage of PTC solu- rate of avoidance between consumer groups was tion consumed, indicating that the sequence in not different. However, most of the intercepts which rams received the PTC solutions did not af- differed across consumer groups (Figures 1 and fect fluid intakes on subsequent test days (Tables 2), which suggests that the point of avoidance as 4 and 5). There was also no sequence effect (P > PTC concentration increases is different between 0.05) on total fluid intake on rest days, which sug- groups. gested the effects of PTC dissipate rapidly (data In trial 1, within the medium and low groups, not shown). Relative to both PTC treatment and no individual consumed more than 5% of the sequence that rams received it on a test day, there highest PTC concentration (12.29  mM) offered. were no treatment or sequence effects (P > 0.05) Because the point at which the greatest avoidance on the amount of water intake during the rest days within the population was observed between the for the morning, afternoon, and total fluid intake. 1.57 and 4.39  mM PTC concentrations in trial 1, The average percentage of water intake on the rest the 12.29  mM concentration was eliminated for days during the morning, afternoon, and total for trial 2.  Furthermore, because the range of PTC the day were 56.8% ± 7.7%, 56.8% ± 3.5%, and concentrations for trial 2 was smaller than that 82.6% ± 3.0%, respectively for trial 1 and 76.6% ± of trial 1, the amount of PTC-solution and water 4.7%, 90.1% ± 3.0%, and 84.3% ± 2.3 %, respect- offered in the morning and the time allotted for in- ively for trial 2. take were increased. These changes made from trial A great deal of variation in PTC-solution intake 1 to trial 2 were to encourage those individuals that was observed between rams (Table 4). In trial 1, the were willing to consume greater concentrations of ram with the greatest intake of PTC consumed 9.7- PTC to differentiate themselves from the popula- fold more PTC than the ram with the lowest intake tion. Although PTC concentrations, total fluid (1.06 vs. 0.109 g, respectively). For trial 2, the mag- offered, and duration that the PTC solution was nitude of difference was much greater at 60-fold available to the rams varied between trials, a similar (2.10 vs. 0.0348 g PTC, respectively). Based on the individual variation in PTC solution intake was still variation between rams within each trial, rams were observed in trial 2 compared with trial 1 (Figures grouped according to total (g) PTC intake (Table 1 and 2). These results suggest that PTC intake is 4). In trial 1, the high intake group consisted of related to the individual ram’s preference for, or three individuals, medium consisted of five, and avoidance to, bitter taste and that PTC can be used Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/3/4/txz125/5581782 by Ed 'DeepDyve' Gillespie user on 18 October 2019 8 Henslee et al. Figure 1. Trial 1 linear regressions of phenylthiocarbamide (PTC) consumption by total PTC intake categories (high: □ − − −; medium: × ——; low: ○ ∙ ∙ ∙ ∙) for Rambouillet and Targhee rams administered five PTC concentrations (0.20, 0.56, 1.57, 4.39, and 12.29 mM) suspended in 1.5 kg of water. Paired contrast made between high vs. low, medium vs. low, and low vs. high. Orthogonal contrast made between high/medium vs. low and high vs. medium/low. Figure 2. Trial 2 linear regressions of phenylthiocarbamide (PTC) consumption by total PTC intake categories (high: □ − − −; medium: × ——; low: ○ ∙ ∙ ∙ ∙) for Rambouillet and Targhee rams administered five PTC concentrations (0.20, 0.43, 0.94, 2.03, and 4.39 mM) suspended in 3.0 kg of water. Paired contrast made between high vs. low, medium vs. low, and low vs. high. Orthogonal contrast made between high/medium vs. low and high vs. medium/low. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/3/4/txz125/5581782 by Ed 'DeepDyve' Gillespie user on 18 October 2019 Phenylthiocarbamide avoidance in rams 9 as a bitter-mimicking agent to determine sensitivity (Sclafani, 1991; Provenza et al., 1992; Launchbaugh to bitterness among individuals. et  al., 2001). Differences in memory of a forage is To date, this is the first study to use PTC to test likely linked to the individual’s physiologic ability for sensitivity of bitterness among sheep. Sheep have to suppress the toxic effects (Provenza et al., 1992). displayed the ability to identify the presence of terpe- Because terpenoids contain bitter-tasting com- nes when fed in a mixed ration (Villalba et al., 2006; pounds, variation in bitter preference between indi- Mote et  al., 2007), which suggests that part of diet viduals may not only translate to forage selection but selection is sensory and is related to taste and(or) may also be correlated with the individual’s ability aroma. Dziba and Provenza (2008) reported that in to suppress toxins. Toxic shrub intake is likely driven lambs offered varying concentrations of monoter- by several phenotypic (Mennella et al., 2005; Dziba penoids (camphor, p-cymeme,1–8 cineole, methacr- et al., 2007; Ginane et al., 2011) and genotypic fac- olein; commonly found in Big Mountain sagebrush) tors (Chandrashekar et al., 2000; Bufe et al., 2005). mixed into their diets, intake rates of the mixed diet in Results from this study indicate that there are relation to monoterpene concentration varied. There sensitivity differences between individual’s preference was no difference in the percentage of time spent eat- for consuming bitter tasting compounds in sheep. ing among the groups (high concentration  =  4.65% The variation in bitterness intake may translate to terpene, medium concentration  =  3.10%, and low foraging preferences while grazing, where rams with concentration = 1.55%), but the medium group con- greater tolerance for bitter taste may consume plants sumed less than the low group and the high group with higher concentrations of bitter tasting com- consumed less than the medium group. Furthermore, pounds, such as monoterpenoids. Similarly, humans Dziba and Provenza (2008) suggested that lambs that are categorized as nontasters consume more regulate feed intake of bitter vegetation to prevent anti-oxidant rich vegetables with bitter attributes consuming a toxic dose of terpenes. Although total than tasters (Garcia-Bailo et al., 2009). amount of forages consumed differed between the Utilizing sheep as a grazing tool to reduce sage- medium and high group, both groups stopped con- brush canopy has been suggested to entail long-term suming feed when they reached approximately 28  g and high-intensity grazing applications (Seefeldt, of monoterpenoids per day (Dziba and Provenza, 2005); however, sheep grazing may be a good tool 2008). Launchbaugh et  al. (1993) observed similar for suppressing sagebrush canopy growth and de- behavior when lithium chloride was mixed in diets crease shrub encroachment on grasslands. Moffet fed to lambs. Despite the concentration at which et  al. (2015) suggested that during a rangeland lithium chloride was fed, lambs regulated their life cycle in a mountain big sagebrush ecosystem, total intake to not exceed concentrations of 62.7  ± the greatest forage productivity and optimal wild- 4.5  mg/kg lithium chloride per day. Regulating in- life habitat conditions occur 5–15  years postfire. take of bitter-tasting compounds is likely a devel- Furthermore, productivity of rangeland decreases oped mechanism that sheep use to avoid forages that as sagebrush canopies become overgrown. Johnson have negative postingestion qualities (Provenza and et  al. (1996) suggested that the greatest ecological Balph, 1987). While postingestion feedback is one diversity in mountain big sagebrush ecosystems oc- mechanism used by ruminants in forage selection, it curs when the sagebrush canopy makes up approxi- is likely not the only deciding factor. mately 15% of total plant composition, and the Launchbaugh (2001) suggested that foraging greatest herbaceous production occurs when the behaviors can be learned from mimicking maternal sagebrush canopy makes up 11–17% of total plant and herd behavior, and taste memory from suck- composition. Because diet selection is moderately ling. Nolte and Provenza (1992) observed that feed- heritable in sheep (h  = 0.28) (Snowder et al., 2001), ing onion-flavored milk to orphan lambs resulted selection for sheep that have a higher tolerance for in a preference for onion-flavored feeds later in life. bitter tasting compounds may translate to sage- Some literature suggests that bitterness is likely not brush canopy growth suppression on rangeland, the apparent causative factor when consuming toxic and therefore, extend the optimal ecological pro- forages, but rather postingestive feedback mech- ductivity-time period beyond 5–15  years postfire anisms (Provenza et  al., 1992; Launchbaugh et  al., (Moffet et al., 2015). 2001). Future selection or determent of a forage is associated with the memory of that taste and the ACKNOWLEDGMENTS digestive feedback; however, memory and toler- ance vary between individuals and each individual The authors would like to acknowledge perceives cost/benefit from a forage differently the support for this research provided by the Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/3/4/txz125/5581782 by Ed 'DeepDyve' Gillespie user on 18 October 2019 10 Henslee et al. Favreau,  A., R.  Baumont, A.  J.  Duncan, and C.  Ginane. USDA-ARS-Sheep Experiment Station employees, 2010. Do sheep use umami and bitter tastes as cues of Mark Williams, Taylor Hudson, and Ashleigh post-ingestive consequences when selecting their diet? Redman. The authors would also like to thank App. Anim. Behav. Sci. 125(3–4):115–123. doi:10.1016/j. 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