We are grateful to the students in the Animal Behavior and Welfare Laboratory at Texas A&#38;M University for their assistance in decoding video recordings of cattle behavior and to the personnel at the Animal Science Teaching and Research Center for their assistance with this project. This project was supported by the Texas A&#38;M University Department of Animal Science Graduate Student Research Mini-Grant program.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Texas A&#38;M University (IACUC2016-0356).
1. Animal and housing
<ol>
	<li>House yearling &#188; Bos indicus x &#190; Bos taurus steers (n = 32) from the same herd in two drylot pens (n = 16 steers/pen) for 7 days prior to test commencement. At the beginning of the study, steers weighed 270.9 &#177; 14.8 kg and were fed the same standard growing ration throughout the study.</li>
	<li>Visually evaluate steers daily as part of routine husbandry practices. No steers received medical treatment throughout the duration of the study.</li>
</ol>
2. Description of the tests
<ol>
	<li>Test 1: Exit velocity
	<ol>
		<li>Place electronic timers in front of a handling chute so that the distance between the starting and stopping points is 1.8 m. These timers are designed to start timing when the animal breaks the first electronic beam and stop when the animal breaks the second electronic beam.</li>
		<li>Move cattle through the handling facility.</li>
		<li>Catch each animal in the headgate of the chute and keep it restrained for 10 s.</li>
		<li>After 10 s, release the animal from the headgate.</li>
		<li>With the electronic timer, record the time it takes for the animal to traverse 1.8 m from the chute.</li>
		<li>Calculate the velocity of the animal as it leaves the chute by dividing 1.8 m by the time it took for the animal to traverse the 1.8 m after release from the headgate and chute. 
		NOTE: Other publications have used this data collection strategy8,9,10,11.</li>
	</ol>
	</li>
	<li>Test 2: Chute score
	<ol>
		<li>Move the cattle through the handling facility.</li>
		<li>Catch each animal in the headgate of the chute for 10 s without applying pressure to its body.</li>
		<li>Have someone observe the cattle for the 10 s and assign each animal a score according to the 2019 Beef Improvement Federation Guidelines for Uniform Beef Improvement Programs 9th Edition (Table 2) based upon its behavior while being restrained.</li>
		<li>After 10 s, release animal from the headgate and chute. 
		NOTE: Other publications have used this data collection strategy12,13,14.</li>
	</ol>
	</li>
	<li>Test 3: Pen score
	<ol>
		<li>Place a group of five cattle in a pen (7.3 m W x 7.3 m L x 2.4 m H).</li>
		<li>Have a single human observer that is unknown to the cattle enter the pen on foot and close the gate after entering the pen.</li>
		<li>Have the observer take two steps towards the group of cattle.</li>
		<li>Visually observe each animal&#39;s behavior in response to the observer.</li>
		<li>Within 30 s of entering the pen, assign each animal a score according to the 2019 Beef Improvement Federation Guidelines for Uniform Beef Improvement Programs 9th Edition (Table 3).</li>
		<li>Clean the testing arena from urine and feces in between groups of animals. 
		NOTE: Other publications that have used this data collection strategy10,11.</li>
	</ol>
	</li>
	<li>Test 4: Bovine zero maze
	<ol>
		<li>Construct a Bovine Zero Maze (BZM).
		<ol>
			<li>Use cattle panels to create a circular track 1.6 m wide, with the inner and outer diameters measuring 6.6 m and 8.2 m, respectively (Figure 1).</li>
			<li>Divide the BZM into four quadrants of equal length with two opposing open quadrants and two opposing closed quadrants where the panels are covered with shade cloth and the shade cloth is stretched across the inner and outer rings of the maze to make a roof over the closed portions of the maze.</li>
			<li>If the test is conducted outdoors, to minimize variation due to shadows, orient the BZM such that the closed sections of the maze face north and south and conduct tests at approximately noon each test day.</li>
		</ol>
		</li>
		<li>Mount a video camera(s) to capture the entire arena. Turn the camera on and begin recording.</li>
		<li>Using low stress handling practices, move a single animal into an open portion of the maze, and allow the animal to explore the arena for 10 min.</li>
		<li>At the end of the 10 min observation period, return the animal to its home pen.</li>
		<li>Clean the testing arena from urine and feces in between animals.</li>
		<li>Decode the video recordings for frequency and latency of steps, escape attempts, kicks, urinations, defecations, vocalizations, standing bouts, duration of time spent standing, duration of time spent walking, latency to enter closed areas, number of times the animal enters closed areas, amount of time in closed/open portions, number of transitions between open/closed arms. Metrics were identified based upon previously published work15.</li>
	</ol>
	</li>
	<li>Test 5: Individual Startle Test and the Group Startle Test
	<ol>
		<li>Construct an arena (7.3 m W x 7.3 m L x 2.4 m H) that has a solid, uniform ground surface free of vegetation or manure, and two closed umbrellas at opposite ends of the arena (Figure 2). The umbrellas should be designed so that they open suddenly at the push of a button.
		<ol>
			<li>Ensure that the sides of the arena are solid or covered with plywood or shadecloth to ensure that the animal cannot see outside of the arena.</li>
			<li>Cut a hole at approximately cattle head height on opposite sides of the arena for the umbrella to penetrate through.</li>
		</ol>
		</li>
		<li>Mount a video camera(s) to capture the entire arena. Turn the video camera on and begin recording.</li>
		<li>Using low stress handling practices, move a single animal into the testing arena. For the group startle test introduce a small group of approximately four animals.</li>
		<li>After the animal(s) has been in the arena for 60 s, open the two umbrellas simultaneously.</li>
		<li>Leave the animal(s) in the arena for 4 min after the umbrellas have opened.</li>
		<li>Clean the testing arena from urine and feces in between tests.</li>
		<li>Decode the video recordings for the frequency and latency of steps, escape attempts, touching the umbrellas, kicks, urinations, defecations, vocalizations, standing bouts, duration of time spent standing, steps in the first 60 s of testing, and steps in the 60 s after the umbrellas were opened for each animal. Metrics were identified based upon previously published work16.</li>
	</ol>
	</li>
	<li>Test 6: Open field test
	<ol>
		<li>Construct a square arena (7.3 m W x 7.3 m L x 2.4 m H) that has a solid, uniform ground surface free of vegetation or manure. The sides of the arena should be solid or covered with plywood or shadecloth to ensure that the animal cannot see outside of the arena.</li>
		<li>Mount a video camera(s) to capture the entire arena. Turn the video camera(s) on and begin recording.</li>
		<li>Using low stress handling practices, move a single animal to the center of a solid sided open field testing arena.</li>
		<li>Leave the animal in the arena for 10 min.</li>
		<li>After 10 min, return the animal to its home pen.</li>
		<li>Clean the testing arena from urine and feces in between animals.</li>
		<li>Decode the video recordings for the frequency and latency to first step, escape attempts, kicks, urination, defecation, vocalization, standing bouts, duration of time spent standing, duration of walking, number of steps taken, number of steps taken during the first 60 s of testing. Metrics were identified based upon previously published work17,18,19.</li>
	</ol>
	</li>
</ol>
3. Statistical analysis
<ol>
	<li>Evaluate inter- and intra-test repeatability using a Pearson&#39;s Correlation (PROC CORR) and reliability calculated using Cronbach&#39;s alpha (PROC CORR). Conduct a validity of response variables with relation to average daily gain (ADG) using a regression analysis (PROC REG).</li>
	<li>After standardizing the variables (PROC STANDARD), use a Cluster Analysis (PROC VARCLUS) to identify relationships among variables from within and among different tests. Many of these variables may be regressed against production metrics to identify production-relevant relationships among cattle behavior during these tests and productivity.</li>
</ol>

<ol>
	<li>Kurvers, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Personality+predicts+the+use+of+social+information.">Personality predicts the use of social information.</a> Ecology Letters. 13 (7), 829-837 (2010).</li><li>St&#246;we, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+object+exploration+in+ravens+(Corvus+corax):+effects+of+social+relationships.">Novel object exploration in ravens (Corvus corax): effects of social relationships.</a> Behavioural processes. 73 (1), 68-75 (2006).</li><li>R&#233;ale, D., Reader, S. M., Sol, D., McDougall, P. T., Dingemanse, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+animal+temperament+within+ecology+and+evolution.">Integrating animal temperament within ecology and evolution.</a> Biological Reviews. 82 (2), 291-318 (2007).</li><li>Sih, A., Bell, A. M., Johnson, J. C., Ziemba, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+syndromes:+an+integrative+overview.">Behavioral syndromes: an integrative overview.</a> The Quarterly Review of Biology. 79 (3), 241-277 (2004).</li><li>Mendl, M., Burman, O. H., Parker, R. M., Paul, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+bias+as+an+indicator+of+animal+emotion+and+welfare:+Emerging+evidence+and+underlying+mechanisms.">Cognitive bias as an indicator of animal emotion and welfare: Emerging evidence and underlying mechanisms.</a> Applied Animal Behaviour Science. 118 (3-4), 161-181 (2009).</li><li>Panksepp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for"+affective+neuroscience"+in+resolving+what+is+basic+about+basic+emotions.">A critical role for" affective neuroscience" in resolving what is basic about basic emotions.</a> Psychological Review. 99 (3), 554-560 (1992).</li><li>Panksepp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emotional+endophenotypes+in+evolutionary+psychiatry.">Emotional endophenotypes in evolutionary psychiatry.</a> Progress in Neuro-Psychopharmacology and Biological Psychiatry. 30 (5), 774-784 (2006).</li><li>Bruno, K., Vanzant, E., Vanzant, K., McLeod, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+of+a+novel+objective+chute+score+and+exit+velocity+with+growth+performance+of+receiving+cattle.">Relationships of a novel objective chute score and exit velocity with growth performance of receiving cattle.</a> Journal of Animal Science. 94 (11), 4819-4831 (2016).</li><li>Burdick, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+exit+velocity+in+suckling+Brahman+calves.">Evolution of exit velocity in suckling Brahman calves.</a> Journal of Animal Science. 89 (1), 233-236 (2011).</li><li>Curley, K., Paschal, J., Welsh, T., Randel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exit+velocity+as+a+measure+of+cattle+temperament+is+repeatable+and+associated+with+serum+concentration+of+cortisol+in+Brahman+bulls.">Exit velocity as a measure of cattle temperament is repeatable and associated with serum concentration of cortisol in Brahman bulls.</a> Journal of Animal Science. 84 (11), 3100-3103 (2006).</li><li>King, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+animal+temperament+and+stress+responsiveness+on+the+carcass+quality+and+beef+tenderness+of+feedlot+cattle.">Influence of animal temperament and stress responsiveness on the carcass quality and beef tenderness of feedlot cattle.</a> Meat Science. 74 (3), 546-556 (2006).</li><li>Hall, N. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Working+chute+behavior+of+feedlot+cattle+can+be+an+indication+of+cattle+temperament+and+beef+carcass+composition+and+quality.">Working chute behavior of feedlot cattle can be an indication of cattle temperament and beef carcass composition and quality.</a> Meat Science. 89 (1), 52-57 (2011).</li><li>MacKay, J., Turner, S., Hyslop, J., Deag, J., Haskell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+temperament+tests+in+beef+cattle+relate+to+long-term+measures+of+behavior+recorded+in+the+home+pen.">Short-term temperament tests in beef cattle relate to long-term measures of behavior recorded in the home pen.</a> Journal of Animal Science. 91 (10), 4917-4924 (2013).</li><li>Voisinet, B., Grandin, T., Tatum, J., O'Connor, S., Struthers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feedlot+cattle+with+calm+temperaments+have+higher+average+daily+gains+than+cattle+with+excitable+temperaments.">Feedlot cattle with calm temperaments have higher average daily gains than cattle with excitable temperaments.</a> Journal of Animal Science. 75 (4), 892-896 (1997).</li><li>Shepherd, J. K., Grewal, S. S., Fletcher, A., Bill, D. J., Dourish, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+and+pharmacological+characterisation+of+the+elevated+"zero-maze"+as+an+animal+model+of+anxiety.">Behavioural and pharmacological characterisation of the elevated "zero-maze" as an animal model of anxiety.</a> Psychopharmacology. 116 (1), 56-64 (1994).</li><li>Forkman, B., Boissy, A., Meunier-Sala&#252;n, M. C., Canali, E., Jones, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+review+of+fear+tests+used+on+cattle,+pigs,+sheep,+poultry+and+horses.">A critical review of fear tests used on cattle, pigs, sheep, poultry and horses.</a> Physiology &amp; Behavior. 92 (3), 340-374 (2007).</li><li>Boivin, X., Le Neindre, P., Chupin, J., Garel, J., Trillat, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+breed+and+early+management+on+ease+of+handling+and+open-field+behaviour+of+cattle.">Influence of breed and early management on ease of handling and open-field behaviour of cattle.</a> Applied Animal Behaviour Science. 32 (4), 313-323 (1992).</li><li>Kilgour, R. J., Melville, G. J., Greenwood, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+differences+in+the+reaction+of+beef+cattle+to+situations+involving+social+isolation,+close+proximity+of+humans,+restraint+and+novelty.">Individual differences in the reaction of beef cattle to situations involving social isolation, close proximity of humans, restraint and novelty.</a> Applied Animal Behaviour Science. 99 (1-2), 21-40 (2006).</li><li>Redbo, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relations+between+oral+stereotypies,+open-field+behavior,+and+pituitary-adrenal+system+in+growing+dairy+cattle.">Relations between oral stereotypies, open-field behavior, and pituitary-adrenal system in growing dairy cattle.</a> Physiology &amp; Behavior. 64 (3), 273-278 (1998).</li><li>Burdick, N., Randel, R., Carroll, J., Welsh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+temperament,+stress,+and+immune+function+in+cattle.">Interactions between temperament, stress, and immune function in cattle.</a> International Journal of Zoology. 2011, (2011).</li><li>Gibbons, J. M., Lawrence, A. B., Haskell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consistency+of+flight+speed+and+response+to+restraint+in+a+crush+in+dairy+cattle.">Consistency of flight speed and response to restraint in a crush in dairy cattle.</a> Applied Animal Behaviour Science. 131 (1-2), 15-20 (2011).</li><li>Mathias, A., Forehand, L., Carstens, G., Daigle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Stress+and+Anxiety:+Development+and+Validation+of+a+Novel+Fear+Test+for+Cattle.">Quantifying Stress and Anxiety: Development and Validation of a Novel Fear Test for Cattle.</a> Journal of Animal Science. 96, 19 (2018).</li><li>Mathias, A., Daigle, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+in+numbers:+Social+isolation+increases+behavioral+responses+of+cattle+during+startle+tests.">Safety in numbers: Social isolation increases behavioral responses of cattle during startle tests.</a> Journal of Animal Science. 97, 18 (2019).</li><li>Lee, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anxiety+influences+attention+bias+but+not+flight+speed+and+crush+score+in+beef+cattle.">Anxiety influences attention bias but not flight speed and crush score in beef cattle.</a> Applied Animal Behaviour Science. 205, 210-215 (2018).</li><li>Voisinet, B., Grandin, T., O'Connor, S., Tatum, J., Deesing, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bos+indicus-cross+feedlot+cattle+with+excitable+temperaments+have+tougher+meat+and+a+higher+incidence+of+borderline+dark+cutters.">Bos indicus-cross feedlot cattle with excitable temperaments have tougher meat and a higher incidence of borderline dark cutters.</a> Meat science. 46 (4), 367-377 (1997).</li><li>Czycholl, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Test-retest+reliability+of+the+Welfare+Quality+animal+welfare+assessment+protocol+for+growing+pigs.">Test-retest reliability of the Welfare Quality animal welfare assessment protocol for growing pigs.</a> Animal Welfare. 25 (4), 447-459 (2016).</li><li>Lawrence, A., Terlouw, E., Illius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+differences+in+behavioural+responses+of+pigs+exposed+to+non-social+and+social+challenges.">Individual differences in behavioural responses of pigs exposed to non-social and social challenges.</a> Applied Animal Behaviour Science. 30 (1-2), 73-86 (1991).</li><li>Panksepp, J., Biven, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The archaeology of mind: Neuroevolutionary origins of human emotions. , (2012).</li><li>Goodman, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperament+affects+rangeland+use+patterns+and+reproductive+performance+of+beef+cows.">Temperament affects rangeland use patterns and reproductive performance of beef cows.</a> Rangelands. 38 (5), 292-296 (2016).</li><li>Walf, A. A., Frye, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+the+elevated+plus+maze+as+an+assay+of+anxiety-related+behavior+in+rodents.">The use of the elevated plus maze as an assay of anxiety-related behavior in rodents.</a> Nature Protocols. 2 (2), 322 (2007).</li><li>Hogg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+validity+and+variability+of+the+elevated+plus-maze+as+an+animal+model+of+anxiety.">A review of the validity and variability of the elevated plus-maze as an animal model of anxiety.</a> Pharmacology Biochemistry and Behavior. 54 (1), 21-30 (1996).</li><li>Janczak, A. M., Andersen, I. L., B&#248;e, K. E., F&#230;revik, G., Bakken, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factor+analysis+of+behaviour+in+the+porcine+and+murine+elevated+plus-maze+models+of+anxiety.">Factor analysis of behaviour in the porcine and murine elevated plus-maze models of anxiety.</a> Applied Animal Behaviour Science. 77 (2), 155-166 (2002).</li><li>Hubbard, A. J., Carstens, G. C., Forehand, L., Daigle, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bovine+Zero+Maze:+Development+of+a+novel+fear+test+for+cattle.">The Bovine Zero Maze: Development of a novel fear test for cattle.</a> Applied Animal Behaviour Science. , 104865 (2019).</li></ol>

The authors have nothing to disclose.

Exit Velocity and Chute Score 
The EV and the CS are both evaluated while the animal is being processed through a handling chute. Although cattle behavior for both the EV and the CS are quantified during the same scenario, behavioral responses to these two tests are not related24. This suggests that the scenario in which the EV (e.g., escaping from restraint) and the CS (e.g., enduring restraint) are assessed may be perceived differently by cattle, and subsequently evaluate different emotional systems. The EV evaluates the behavior of cattle as they are escaping from restraint and is therefore thought to evaluate the FEAR system while the CS may evaluate RAGE. The CS evaluates the behavior of cattle while being restrained in the handling chute (Table 2), and thus may be a good proxy for the RAGE emotional system.
Substantial research has been conducted on the relationship between EV and production, health, and behavioral traits. While EV can be influenced by an animal&#39;s previous experience, this objective metric may be effective in quantifying the FEAR system, as substantial relationships between EV and health, productivity, breeding, and behavior have been identified. Cattle with faster EV have reduced growth rates14, poor carcass quality11,25, reduced immune function20, and higher cortisol levels during handling10. This measurement can provide information about behavior in the home pen, because EV is positively correlated with step counts in the home pen13. From an animal management perspective, cattle with faster EV are more difficult to handle, present greater risk to animal managers, and may influence the behavior of herd-mates. While EV may be a good metric for evaluating FEAR, it does not measure all emotional systems. Therefore, additional tests are required to evaluate all of the emotional systems influencing production and welfare.
Pen Score 
The PS subjectively evaluates the cattle&#39;s willingness to be approached by a human (Table 3) and may be useful in evaluating the PANIC system. However, the PS has been criticized for lack of objectivity, because different evaluators may have different interpretations of behavior, and several subjective evaluations have suffered from poor inter-rater reliability26.
Startle Test 
Anxiety is highly evolved in all prey species. High levels of FEAR help protect the animal from pain and activates the sympatho-adrenal and hypothalamic-pituitary-adrenal axes as part of the fight or flight and stress response to a perceived danger. The startle test evaluates an animal&#39;s response to sudden, novel stimuli, and has been identified as an effective measurement in identifying behavioral differences among different genetic strains of pigs27. The startle test may be effective in evaluating the sensitivity and reactivity of the sympatho-adrenal system, which has production-relevant consequences when activated and may provide insight into the FEAR system.
Open Field Test 
The OFT is the most commonly used test. The OFT was originally designed to evaluate individual animal boldness, or willingness to enter an open arena, an environment that may be perceived as dangerous and risky to the animal&#39;s survival. The OFT has been validated for species that instinctively seek shelter and avoid open spaces, such as rodents, chickens, and turkeys16.
Cattle evolved to live in open fields, thus the OFT may not induce the behavioral and physiological responses associated with FEAR and may be better suited to evaluate social isolation (PANIC/GRIEF) or exploration (SEEKING). Further, the OFT evaluates individual animals, and because cattle are gregarious herd animals, the experience of the OFT may be eliciting an emotional response other than FEAR. The OFT lacks a strong correlation with other FEAR tests and the results are difficult to interpret (i.e., many factors can lead to the same activity). Therefore, the OFT is not recommended as a general FEAR test for cattle16 and may not provide a comprehensive understanding of the FEAR systems in cattle. The OFT may, however, be a useful tool in quantifying either the PANIC or the SEEKING systems in cattle.
The SEEKING system is essential for animals to acquire the resources needed for survival. High SEEKING levels provoke intense, persistent enthusiastic exploration, appetitive and anticipatory excitement, and learning. This system can result in forward locomotion as the animal is motivated to explore its surroundings. SEEKING can play a role in both positive and negative emotions; positive SEEKING may engender a sense of purpose while negative SEEKING may result in behaviors associated with safety28. Cows that spent more time exploring and explored a larger portion of the range (e.g., stronger activation of SEEKING) ate quicker while in confinement, had calves with heavier weaning weights, higher cortisol concentrations during confinement, and shorter postpartum intervals to estrus29. Therefore, the SEEKING system can have production and welfare implications. Identifying animals with high activation of the SEEKING system may be more successful in extensive ranging environments where individual and reproductive fitness is dependent on the animal&#39;s capacity to find resources and shelter. However, animals with high activation of the SEEKING system may experience higher levels of stress and frustration during confinement.
Bovine Zero Maze 
Commonly used tests in biomedical research that are designed to evaluate the efficacy of anti-anxiety and anti-depressant drug development in rodents are the elevated plus maze (EPM) and the elevated zero maze (EZM)30. These tests exploit the instinctual behavior of the rodent and its natural propensity for dark, closed-in places to quantify their willingness to explore environments that would be inherently fearful or induce anxiety. Metrics from these tests can include the latency to leave the darkened arm of the maze, duration of time in the open and closed arms of the maze, and the number of transitions between the two environments during the testing period as well as the behavior of the animal (e.g., vocalization, urination, defecation, escape attempts) during the test31.
The EPM and the EZM are both well validated tests for quantifying FEAR/ANXIETY in rodents15,31. A modified EPM has been used to quantify the FEAR response in swine32 but has not been utilized in ruminants. However, the EPM has been criticized for its ambiguity of interpretation regarding behavior in the central square of the maze. Therefore, the EZM was designed to evaluate the same metrics as the EPM but allows uninterrupted exploration without ambiguity. When identifying a test to evaluate FEAR/ANXIETY and SEEKING in cattle, the EZM was a logical model. The EZM is conducive to the natural behavior of cattle, as they instinctively move in circular patterns and have a propensity for returning to the areas from which they came.
By applying principles similar to the EZM with an inverse of interpretation, a Bovine Zero Maze33 has been developed to evaluate the FEAR, PANIC/GRIEF, and SEEKING systems in cattle. Cattle evolved to live in open spaces; therefore, cattle with reduced activation of the FEAR and PANIC/GRIEF systems will be more willing to spend time in the open portions of the BZM than the darkened portions of the maze, will be less likely to enter the closed portions of the maze, and will perform more escape attempts.
Quantifying cattle behavior across multiple evaluations may identify complex emotional relationships that can have economic significance, are easily measured, and can be included in breeding selection efforts. The emotional circuits of PLAY, LUST, and CARE were not evaluated in this study.

Animal temperament has been linked to behavioral characteristics such as exploratory behavior and boldness1,2 and can exhibit consistencies over time and across contexts3,4. However, temperament is composed of multiple emotional systems working together. Animals experience physical and psychological stressors, and evaluating the emotional response to both types is challenging. Emotional state can influence how animals perceive stimuli (e.g., cognitive bias), and is a critical component of animal welfare5. Understanding how an individual will behave in response to psychological stressors (e.g., commingling, weaning, change in stockperson) will provide animal managers additional selection criteria when identifying animals that have the skills to cope with psychological stressors.
Emotions are controlled by seven core affective systems within the brain (Table 1)6. These systems include four that control positive emotions: 1) SEEKING (exploration), 2) LUST (sexual excitement), 3) CARE (nurturance), and 4) PLAY (social joy). Three systems control negative emotions 1) FEAR (anxiety), 2) RAGE (anger), and 3) PANIC/GRIEF (separation distress). These affective systems may be heritable7, impact profitability, and are a critical component of animal welfare.
A battery of tests has been developed to evaluate cattle temperament (e.g., exit velocity, chute score). However, the evaluation of temperament has the potential to be influenced by current conditions, previous experiences, and observer bias. While many of these behavioral evaluations are commonly referred to as fear tests, they may be quantifying different emotional components of temperament other than FEAR. In addition, the variation in how these tests have been conducted makes comparisons across different evaluations challenging. Thus, there is a need to understand the relationships among these behavioral evaluations as well as have a standardized protocol for these temperament evaluations.
The goal of this article is to visually document the different fear tests used for cattle; present the type of data that were generated from these different tests; evaluate the repeatability, validity, and reliability of these tests; demonstrate how to evaluate the relationships among the behaviors captured from these tests; and suggest which emotional circuit could be evaluated with each test.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Electronic timers</td>
			<td>FarmTek, Wylie, TX</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Priefert Cattle Panels</td>
			<td>Priefert Rodeo &#38; Ranch Equipment, Mount Pleasant, TX, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shade Cloth</td>
			<td>Windscreen4less, San Bernardino, CA, USA</td>
			<td>Heavy Duty Privacy Screen Fence in Color Solid Black</td>
			<td></td>
		</tr>
		<tr>
			<td>SILENCER Commerical Pro</td>
			<td>Silencer Hydraulic Chutes, Stapleton, NE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Umbrella</td>
			<td>WinCraft</td>
			<td>Model# A04852, Winona, Minnesota</td>
			<td></td>
		</tr>
		<tr>
			<td>Video Camera</td>
			<td>Canon</td>
			<td>Canon VIXIA HF R800 HD, Mellville, NY, USA</td>
			<td></td>
		</tr>
	</tbody>
</table>

the use of traditional fear tests to evaluate different emotional circuits in cattle

Animal temperament is complex and has implications for productivity and economic profitability. Quantifying an animal&#39;s response to differing stimuli may facilitate breeding selections and identify animals that are better suited to specific management strategies. Multiple tests have been developed to evaluate cattle temperament (e.g., exit velocity, chute score, pen score, open field test, startle test, bovine zero maze), but each of these tests evaluates the animal&#39;s response to different stimuli (e.g., isolation, novel environment, startle, willingness to enter an enclosed area). Cattle temperament has been observed to be relatively stable over time. However, the evaluation of temperament has the potential to be influenced by current conditions, previous experiences, and observer bias. Many of these temperament tests have been improperly categorized as fear tests and have also been criticized for being subjective. This paper provides a framework for standardizing behavioral tests for cattle and suggests that these different evaluations assess different aspects of the animal&#39;s overall temperament.

Watch this Scientific Journal Video about The Use of Traditional Fear Tests to Evaluate Different Emotional Circuits in Cattle at JoVE.com

The Use of Traditional Fear Tests to Evaluate Different Emotional Circuits in Cattle

Here, we present a protocol to conduct a variety of behavioral tests in cattle that have been designed to evaluate emotions. A battery of behavioral tests (open field test, startle test, bovine zero maze, exit velocity, pen score, and chute score) were conducted to evaluate different components of animal temperament.

Animal temperament is complex and has implications for productivity and economic profitability. Quantifying an animal&#39;s response to differing stimuli ...

the-use-traditional-fear-tests-to-evaluate-different-emotional

Research

JoVE Journal

Behavior

8.4K Views.  Texas A&M University. Here, we present a protocol to conduct a variety of behavioral tests in cattle that have been designed to evaluate emotions. A battery of behavioral tests (open field test, startle test, bovine zero maze, exit velocity, pen score, and chute score) were conducted to evaluate different components of animal temperament.

Video: The Use of Traditional Fear Tests to Evaluate Different Emotional Circuits in Cattle

Using Eye Movements to Evaluate the Cognitive Processes Involved in Text Comprehension

The present article describes how to use eye tracking methodologies to study the cognitive processes involved in text comprehension. Measuring eye movements during reading is one of the most precise methods for measuring moment-by-moment (online) processing demands during text comprehension. Cognitive processing demands are reflected by several aspects of eye movement behavior, such as fixation duration, number of fixations, and number of regressions (returning to prior parts of a text). Important properties of eye tracking equipment that researchers need to consider are described, including how frequently the eye position is measured (sampling rate), accuracy of determining eye position, how much head movement is allowed, and ease of use. Also described are properties of stimuli that influence eye movements that need to be controlled in studies of text comprehension, such as the position, frequency, and length of target words. Procedural recommendations related to preparing the participant, setting up and calibrating the equipment, and running a study are given. Representative results are presented to illustrate how data can be evaluated. Although the methodology is described in terms of reading comprehension, much of the information presented can be applied to any study in which participants read verbal stimuli.

The present article describes how to use eye tracking methodologies to study the cognitive processes involved in text comprehension. Descriptions of eye tracking equipment, how to develop experimental stimuli, and procedural recommendations are included. The information presented can be applied to most any study using verbal stimuli.

The present article describes how to use eye  tracking methodologies to study the cognitive processes involved in text  comprehension. Measuring eye ...

An Alternative to the Traditional Cold Pressor Test: The Cold Pressor Arm Wrap

Recently research on the relationship between stress and cognition, emotion, and behavior has greatly increased. These advances have yielded insights into important questions ranging from the nature of stress&#39; influence on addiction1 to the role of stress in neural changes associated with alterations in decision-making2,3. As topics being examined by the field evolve, however, so too must the methodologies involved. In this article a practical and effective alternative to a classic stress induction technique, the cold pressor test (CPT), is presented: the cold pressor arm wrap (CPAW). CPT typically involves immersion of a participant&#39;s dominant hand in ice-cold water for a period of time4. The technique is associated with robust activation of the sympatho-adrenomedullary (SAM) axis (and release of catecholamines; e.g.&#160;adrenaline and noradrenaline) and mild-to-moderate activation of the hypothalamic-pituitary-adrenal (HPA) axis with associated glucocorticoid (e.g.&#160;cortisol) release. While CPT has been used in a wide range of studies, it can be impractical to apply in some research environments. For example use of water during, rather than prior to, magnetic resonance imaging (MRI) has the potential to damage sensitive and expensive equipment or interfere with acquisition of MRI signal. The CPAW is a practical and effective alternative to the traditional CPT. Composed of a versatile list of inexpensive and easily acquired components, CPAW makes use of MRI-safe gelpacs cooled to a temperature similar to CPT rather than actual water. Importantly CPAW is associated with levels of SAM and HPA activation comparable to CPT, and can easily be applied in a variety of research contexts. While it is important to maintain specific safety protocols when using the technique, these are easy to implement if planned for. Creation and use of the CPAW will be discussed.

The cold pressor test is a classic stress induction task used in a wide range of research examining the relationship between stress and cognition, emotion, and behavior. In this article, a practical and versatile alternative to the traditional cold pressor test is presented: the cold pressor arm wrap.

Recently research on the relationship between stress and cognition, emotion, and behavior has greatly increased. These advances have yielded insights into ...

Trace Fear Conditioning in Mice

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings between a neutral stimulus and an unconditioned stimulus. There is a 20 sec trace period that separates each conditioning trial. On the following day freezing is measured during presentation of the conditioned stimulus (CS) and trace period. On the third day there is an 8 min test to measure contextual memory. The representative results are from mice that were presented with the aversive unconditioned stimulus (shock) compared to mice that received the tone presentations without the unconditioned stimulus. Trace fear conditioning has been successfully used to detect subtle learning and memory deficits and enhancements in mice that are not found with other fear conditioning methods. This type of fear conditioning is believed to be dependent upon connections between the medial prefrontal cortex and the hippocampus. One current controversy is whether this method is believed to be amygdala-independent. Therefore, other fear conditioning testing is needed to examine amygdala-dependent learning and memory effects, such as through the delay fear conditioning.

In the following experiment we describe a protocol for trace fear conditioning in mice. This type of associative memory includes a trace period that separates the neutral stimulus and the unconditioned stimulus.

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings ...

Using the Threat Probability Task to Assess Anxiety and Fear During Uncertain and Certain Threat

Fear of certain threat and anxiety about uncertain threat are distinct emotions with unique behavioral, cognitive-attentional, and neuroanatomical components. Both anxiety and fear can be studied in the laboratory by measuring the potentiation of the startle reflex. The startle reflex is a defensive reflex that is potentiated when an organism is threatened and the need for defense is high. The startle reflex is assessed via electromyography (EMG) in the orbicularis oculi muscle elicited by brief, intense, bursts of acoustic white noise (i.e., &#8220;startle probes&#8221;). Startle potentiation is calculated as the increase in startle response magnitude during presentation of sets of visual threat cues that signal delivery of mild electric shock relative to sets of matched cues that signal the absence of shock (no-threat cues). In the Threat Probability Task, fear is measured via startle potentiation to high probability (100% cue-contingent shock; certain) threat cues whereas anxiety is measured via startle potentiation to low probability (20% cue-contingent shock; uncertain) threat cues. Measurement of startle potentiation during the Threat Probability Task provides an objective and easily implemented alternative to assessment of negative affect via self-report or other methods (e.g., neuroimaging) that may be inappropriate or impractical for some researchers. Startle potentiation has been studied rigorously in both animals (e.g., rodents, non-human primates) and humans which facilitates animal-to-human translational research. Startle potentiation during certain and uncertain threat provides an objective measure of negative affective and distinct emotional states (fear, anxiety) to use in research on psychopathology, substance use/abuse and broadly in affective science. As such, it has been used extensively by clinical scientists interested in psychopathology etiology and by affective scientists interested in individual differences in emotion.

Potentiation of the startle reflex is measured via electromyography of the orbicularis oculi muscle during low (uncertain) and high (certain) probability electric shock threat in the Threat Probability Task. This provides an objective measure of distinct negative emotional states (fear/anxiety) for research on psychopathology, substance use/abuse, and broad affective science.

Fear of certain threat and anxiety about uncertain threat are distinct emotions with unique behavioral, cognitive-attentional, and neuroanatomical ...

The Emotional Stroop Task: Assessing Cognitive Performance under Exposure to Emotional Content

The emotional Stroop effect (ESE) is the result of longer naming latencies to ink colors of emotion words than to ink colors of neutral words. The difference shows that people are affected by the emotional content conveyed by the carrier words even though they are irrelevant to the color-naming task at hand. The ESE has been widely deployed with patient populations, as well as with non-selected populations, because the emotion words can be selected to match the tested pathology. The ESE is a powerful tool, yet it is vulnerable to various threats to its validity. This report refers to potential sources of confounding and includes a modal experiment that provides the means to control for them. The most prevalent threat to the validity of existing ESE studies is sustained effects and habituation wrought about by repeated exposure to emotion stimuli. Consequently, the order of exposure to emotion and neutral stimuli is of utmost importance. We show that in the standard design, only one specific order produces the ESE.

The emotional Stroop effect (ESE) is the result of longer naming latencies to ink colors of emotion words than those of neutral words. This report refers to potential sources of confounding and includes a modal experiment that provides the means to control for them.

The emotional Stroop effect (ESE) is the result of longer naming latencies to ink colors of emotion words than to ink colors of neutral words. The ...

Exploring the Use of Isolated Expressions and Film Clips to Evaluate Emotion Recognition by People with Traumatic Brain Injury

The current study presented 60 people with traumatic brain injury (TBI) and 60 controls with isolated facial emotion expressions, isolated vocal emotion expressions, and multimodal (i.e., film clips) stimuli that included contextual cues. All stimuli were presented via computer. Participants were required to indicate how the person in each stimulus was feeling using a forced-choice format. Additionally, for the film clips, participants had to indicate how they felt in response to the stimulus, and the level of intensity with which they experienced that emotion.

This paper describes how to implement a battery of behavioral tasks to examine emotion recognition of isolated facial and vocal emotion expressions, and a novel task using commercial television and film clips to assess multimodal emotion recognition that includes contextual cues.

The current study presented 60 people with traumatic brain injury (TBI) and 60 controls with isolated facial emotion expressions, isolated vocal emotion ...

A Protocol of Manual Tests to Measure Sensation and Pain in Humans

Numerous qualitative and quantitative techniques can be used to test sensory nerves and pain in both research and clinical settings. The current study demonstrates a quantitative sensory testing protocol using techniques to measure tactile sensation and pain threshold for pressure and heat using portable and easily accessed equipment. These techniques and equipment are ideal for new laboratories and clinics where cost is a concern or a limiting factor. We demonstrate measurement techniques for the following: cutaneous mechanical sensitivity on the arms and legs (von-Frey filaments), radiant and contact heat sensitivity (with both threshold and qualitative assessments using the Visual Analog Scale (VAS)), and mechanical pressure sensitivity (algometer, with both threshold and the VAS). The techniques and equipment described and demonstrated here can be easily purchased, stored, and transported by most clinics and research laboratories around the world. A limitation of this approach is a lack of automation or computer control. Thus, these processes can be more labor intensive in terms of personnel training and data recording than the more sophisticated equipment. We provide a set of reliability data for the demonstrated techniques. From our description, a new laboratory should be able to set up and run these tests and to develop their own internal reliability data.

The goal of this procedure is to demonstrate a battery of quantitative techniques for sensory and pain measurement in humans. The equipment and techniques described are commonly found in pain clinics or are easy to obtain.

Numerous qualitative and quantitative techniques can be used to test sensory nerves and pain in both research and clinical settings. The current study ...

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often stereotyped actions elicited in response to innately aversive sensory stimuli, but their success requires enough flexibility for adapting to different spatial environments, which can change rapidly. Here, we describe a behavioral assay to evaluate the influence of learned spatial knowledge on defensive behaviors in mice. We have adapted the widely used Barnes maze spatial memory assay to investigate how mice navigate to a shelter during escape responses to innately aversive sensory stimuli in a novel environment, and how they adapt to acute changes in the environment. This new assay is an ethological paradigm that does not require training and exploits the natural exploration patterns and navigation strategies in mice. We propose that the set of protocols described here are a powerful means of studying goal-directed behaviors and stimulus-triggered navigation, which should be of interest to both the fields of instinctive behaviors and spatial memory.

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often ...

The Adventures of Fundi Intervention Based on the Cognitive and Emotional Processing in Attention Deficit Hyperactive Disorder Patients

Many children with attention deficit hyperactive disorder (ADHD) are known to have executive dysfunction, which weakens their abilities to learn and behave in daily living. This protocol describes the methodology that is required for the intervention (psychotherapy) based on planning, attention, successive, and simultaneous (PASS theory) cognitive processing and fear emotional processing. It provides guiding principles and practical recommendations. A disproportionately high level of fear (dysregulation) increases the vulnerability for dysfunction in learning and behavior. We explain the interplay between emotion and cognition at the neurological level.
A go/no go task (The Adventures of Fundi), which involves decision making, is administered in a PC- mode to a sample of 66 ADHD subjects. The Adventures of Fundi, a computer program, was constructed to induce successive or simultaneous processing when involving the training of planning and selective attention. It aims to improve the executive function with planning and selective attention. If executive function improves, learning improves, and behavior ameliorates. After intervention over 6 months, remission was achieved in 70% of subjects.
The instructor encourages the use of appropriate strategies and points out the ways in which the strategies can be useful in finding the solution to the problem (go/no go). The emphasis is not on rehearsing and adult instructed verbal sequence. The verbalization may reveal the conscious verbalized strategy to solve a task that is not really the strategy being unconsciously used in that case. A self-verbal report is unreliable. This is an inductive learning rather than deductive rule-learning approach central to cognitive PASS training. This inductive training has proved to produce not only near transfer but also far transfer.
Noncognitive factors (emotional factors) must be considered to maximize the benefit of cognitive training. Indirect and metaphorical communication considers the emotional factor.

This protocol illustrates a remedial therapy based on inductive learning and indirect communication (Ericksonian metaphorical hypnosis) that can be applied to children with Attention Deficit Hyperactive Disorder (ADHD). Particularly, this protocol is intended to test the effectiveness of a go/no-go decision task. Neuroscientific rationale is presented throughout the text.

Many children with attention deficit hyperactive disorder (ADHD) are known to have executive dysfunction, which weakens their abilities to learn and ...

Examining Changes in HRV and Emotion Following Artmaking with Three Different Art Materials

This protocol enables the examination of psychological and physiological responses to different types of behavioral engagements. Specifically, in this study example, the emotional response and changes in heart rate variability are examined in response to artmaking with three different art materials that vary in their levels of fluidity. This protocol can be adapted to examine other types of behavior or engagement in artmaking with other materials. There are several benefits to using this protocol. Firstly, the order randomization of the materials improves the probability that the response measured is associated with its qualities and not the order of presentation. Secondly, the continuous measuring of electrocardiogram enables the examination of changes in heart rate variability after engagement with each art material and changes that might occur during the artmaking itself. The advantages of this protocol should be considered with their limitations. The music listening is before each art making session; thus, the return to baseline can only be measured in the first two conditions. The return to baseline provides information on how fast individuals recover after response to working with each of the materials. Furthermore, a more liquid material instead of gouache paint with a brush, such as finger paints, provides more difference between materials. Finally, this protocol can be adapted to specific research needs.

The goal of the protocol is to guide researchers in conducting experiments that are intended to measure changes in self-reported emotional response and heart rate variability following art making with different materials. The protocol can easily be adapted for use in a variety of behavioral conditions and activities.

This protocol enables the examination of psychological and physiological responses to different types of behavioral engagements. Specifically, in this ...