The authors would like to acknowledge funding from the Office of Naval Research (Grant #12166253). In addition, the authors kindly thank the animal care staff, veterinarians, and students at Kansas State University and Virginia Tech for their support during the animal work. The authors would also like to thank Nad&#232;ge Krebs for her technical assistance, and students Shelby Stair, Sarah Greenway, and Mikayla Goering for their technical assistance and additional animal care.

The individual IACUC at each location (Kansas State University and Virginia Tech University) approved the use of facilities and procedures.
1. Set-up of the Cameras and Pens and the Establishment of a Routine
<ol>
	<li>Prior to placing the animals in their pens, fix cameras at a 90&#176; angle over each pen (see Table of Materials for a suggested camera system).</li>
	<li>Record animals continuously at 30 frames/s (fps), either for the duration of the study or only during the test sessions.</li>
	<li>Fix bowls, waterers, mats, and toys with bolts and chains.</li>
	<li>Place sound machines that continuously play white or pink noise (e.g., the sound of waterfalls) in the pigs&#8217; facility. 
	NOTE: External sudden noises (e.g., doors opening and shutting) can cause a startle reflex during the sessions23,24.</li>
	<li>Randomize or stratify treatments by pen across the facility.</li>
	<li>Set up an established animal husbandry routine. This will help the pigs to know when to expect humans to clean the pens, feed and handle them, and perform the test.
	<ol>
		<li>Use a familiar treat to allow the pigs to associate humans with a reward.</li>
		<li>Use a clicker during the reward to allow the pigs to associate the clicking sound with a reward. Do not use vocalizations and visual signals to familiarize the pigs with the reward (see Table of Materials).</li>
	</ol>
	</li>
	<li>Conduct the sessions before the morning meal or before placing a new feed for ad libitum fed pigs.</li>
</ol>
2. Identification of the Pigs
<ol>
	<li>Make subjects identifiable on the video feed, even if they are single-housed.</li>
	<li>Make sure observers remain blind to treatments and unbiased during timestamping with a marking system that is not related to treatments. Use a medical grade tape (see Table of Materials) that is adhered to a strip of duct tape of a specific color, round shape, and pattern.</li>
	<li>Use one round patch to mark the top of the pig and one down each side (see Figure 1, green and blue markers).</li>
	<li>Smear tag cement (less than 0.35 g) on the tape corners to help increase the adherence longevity. 
	NOTE: Too much tag cement will not dry very quickly, causing the tape to fall off prematurely.</li>
	<li>Troubleshoot and customize the marking strategy during the acclimation period so that official tests are performed efficiently and without added stress to the pigs.</li>
	<li>Use data collection sheets to keep track of the markings and subject identification (see Supplementary File 1 for an example data sheet). 
	NOTE: To remove tape, do not rip the tape off because it will cause pain if any hair is pulled out. It can either slough off on its own, a water-based lubricant can help dislodge it, or it can be shaved off with clippers. If the tape comes off at unwanted times, prepare extra marking tape and reapply it while the pig is eating a meal rather than restraining the pig.</li>
</ol>
3. HAT Sessions
<ol>
	<li>Have the test-humans wear the same hat, coveralls, boots, odors, etc. every time the session is conducted.</li>
	<li>Conduct sessions daily, at least 3x, before the treatment and, then, daily thereafter.</li>
	<li>The pig can be in any area in its pen before starting the session. To start the session, ask the test-human to drop the treat in the bowl or in front of the pen and click the clicker 3x.</li>
	<li>The test-human needs to place their hands out of the pig&#8217;s sight and stand stationary during the test. Have another researcher mark the start of the session on the data sheet and start a timer. After 120 s, the researcher silently signals to the test-human to move to the next subject and restart the test.</li>
</ol>
4. Establishment of HAT Ethograms for Software
<ol>
	<li>Construct ethograms (see Figures 1 and 2 and Supplementary Video 1) under one project using specialized software. 
	NOTE: Spatial behaviors are the location of the animal relative to the human. In the ethograms, the spatial relationships must be customized to the animal&#8217;s pen and published every time a novel pen set-up is used (Figure 1). Within this category, behaviors are considered mutually exclusive. The space is divided into four areas, with varying levels of approach (Figure 1). The areas are standardized across housing types. Closest, or climb (Cl) means that pigs may climb on the fence to gain access to the human; therefore, climbing is considered a spatial behavior that indicates the pig is most actively seeking human contact. Close (Co) denotes the area within 61 cm of the human. Mid (M) is the area within 61-122 cm from the human. Far (F) is the area of 123 cm or more from the human.</li>
	<li>Structural behaviors focus on the orientation of the whole body or parts of the body (Figure 2). Create two structural categories: 1) the pig-nose position and 2) the active state of the pig. Use Figure 2A to identify directionality (direction 1 denotes the pig directs its nose towards the human; direction 2 means the pig directs its nose away from the human).</li>
	<li>Divide activity behaviors (Figure 2B) into three mutually exclusive states of approach: NNOB, stand or walk without NNOB, and resting without NNOB (see the color coding in the ethograms in the video). 
	NOTE: NNOB is used to describe when a pig is using its mouth, snout, or face to lick, sniff, chew, bite, rub, or root a nonnutritive object to seek out either familiarity or novel opportunities. Therefore, when it is in this active state, it is interested in the human, and in an approach-state. If overhead cameras are used at 90&#176; angles, the pig&#8217;s nose position and head movement are indicators of NNOB. Occasionally, pigs will chomp or sham-chew; the nose can be seen but the head moves up and down. Stand or walk without NNOB is used to the describe when the pig is in the upright position, the head is still, and the nose is not touching a substrate or chomping, which means it is in less of an approach state. Resting without NNOB is used the describe when the pig is resting by lying or sitting, which is the least approach state under this category of behaviors.</li>
</ol>
5. Timestamping of the Videos for Efficiency and Reliability
<ol>
	<li>Based on the start-times recorded by the data collector, edit the footage into exact, 3 min sessions. The method for timestamping will take 9 min per session.</li>
	<li>Only use up to two trained observers to timestamp the videos. 
	NOTE:: If two observers are used, the intra-observer variation should be quantified, evaluated, adjusted, and then reported as a Pearson correlation coefficient after the observers have timestamped the same sample video (for methods, see Martin and Bateson25).</li>
	<li>Set the playback speed at 1x regular speed (i.e., 30 fps). Do not pause, rewind, or timestamp frame by frame.</li>
	<li>Timestamp each category of mutually exclusive behaviors separately.
	<ol>
		<li>Timestamp spatial behaviors. Restart the video.</li>
		<li>Timestamp structural behaviors. Restart the video.</li>
		<li>Timestamp for structural/nose position behaviors.</li>
	</ol>
	</li>
	<li>Use the duration of each behavior outcome (see Supplementary Videos 2 and 3) for summarizing data. The duration measurements need to be converted to a percentage of time per category.</li>
</ol>
6. Approach Index
<ol>
	<li>Apply the formula (Figure 3) so that each structural and the spatial behavior is combined to create an AI (Figure 3, Figure 4). The AI is used in addition to reporting behaviors and categories separately as figures (Figure 5) or in tabular form. 
	NOTE: See Table of Materials for software details. Within each category, behavior durations are first converted to percentages (the duration of the behaviors divided by the total duration of the test session). Each behavior is weighted based on the level of approach (Figure 3). Avoidance behaviors (the percentage of time in the far area, resting, with the nose turned away) are multiplied by 0. Moderate-approach behaviors (the percentage of time in the middle area, turned towards the human, and standing with the head still) are multiplied by 1. The greatest level of approach behaviors (the percentage of time in the close and closest area and showing NNOB) are multiplied by 2. Then, each category is further weighted 3, 2, and 1 for spatial, nose orientation, and activity, respectively. A constant (0.10) is applied to create a percentage scale. For example, if the pig faces the human, is in the close/closest area, and performs NNOB during the entire test session, note that the AI is 100% (the red pig in Figure 3). In contrast, if the pig is in the far area, is not facing the human, and remains in the rest position during the entire test session, the AI is 0% (the black pig in Figure 3). Pigs in the close zone can have the same AI as a pig performing NNOB in the mid area if they are turned away from the human and standing with their head still (the orange pigs in Figure 3).</li>
</ol>

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The authors have nothing to disclose.

Mild injuries to the brain that do not result in overt anatomical and structural changes detectable with state-of-the-art imaging can be difficult to identify and treat28. However, patients with mTBI are especially vulnerable to additional insult that can cause significant damage to the brain, and therefore, it is important for this population to be identified. Behavioral tests developed in a mini-pig model of mTBI are especially relevant to human mTBI patients because pigs have a similar physiology as humans and express similar affective states, such as anhedonia8,9,10,20. Here, we have developed a noninvasive, in-pen behavioral test (the HAT), and have shown that it is sensitive enough to distinguish mTBI pigs from sham pigs. In addition, a weighted index (the AI) was developed for behaviors observed during the HAT that are ubiquitous across housing and pig types.
Modifications and Troubleshooting:
The methodologies for the HAT were established based on ethology guidelines25 and several trial-and-error strategies for improving the reliability, repeatability, and validity of the test6. Reliability measures helped identify the strengths and limitations of the test. Reliability defines the extent to which the measurement is repeatable and consistent and free from random errors28,29. We have previously reported on the intra- and interobserver reliability of the HAT, and with the additional structural ethograms, reliabilities are similarly high (Pearson&#39;s R2 &#62; 0.90) for duration6. Frequency and latency measures require trained observers, whereas duration measures are less observer-dependent, and therefore, more reliable across laboratories30.
Reliability within a laboratory and repeatability across laboratories is dependent on methods. In our laboratory, the video system recorded continuously, the files were initially stored as 5-minute files, and some HAT sessions occurred over two files. Fewer mistakes were made when the exact time from the data sheet was used to clip and combine the videos. Before developing the ethogram, observers were allowed to pause, stop, and rewind the video footage to timestamp all of the behaviors in the entire ethogram. This method not only caused variation in timestamping each sample, ranging from 3 min to 20 min, but the between- and within-observer reliability was also poor for most behaviors. Therefore, we set the playback speed, and had observers timestamp each category at a time. Therefore, when reliability was low in just one category, observers independently retimestamped just the category rather than the entire ethogram, after they consulted the definitions and footage together. The set playback and category methods allowed for a consistent prediction of how much time was needed to timestamp each sample. For projects that span longer than a month, routine reviewing of the coded videos and within-observer reliability is important to measure.
Another factor that reduces reliability and repeatability is the video set-up. Initially, a handheld camera and a tripod were used, which were moved from pen to pen. When this method was used, the pigs needed to be introduced to the tripod and camera before the HAT; otherwise, the pigs appeared to react to the tripod and movement more than to the test-human. In addition, nonoverhead camera angles limited the observer&#39;s view during timestamping and the depth perception of space increased the within- and between-observer variation in the spatial behavior measures; therefore, we developed the protocol with fixed cameras. When this method is used, extra care is needed to make sure the camera is placed correctly before each test, and more time is needed for the set-up in between each pig&#39;s session. However, we learned that the continuous video overhead system needed to start its initial recording at midnight at least 24 h before the first HAT. The timestamp display for many video systems are not accurate and in sync down to the frame; therefore, we no longer rely on the display times. The midnight starts allowed for the exact frame-capturing and video editing, and the timestamp display was not used.
In addition, the acclimation of the pigs and setting up a routine was important when troubleshooting this test. In footage of pigs that were not well acclimated to their environments, pacing was observed during the HAT. This is an indicator that the pig may be in an agitated state31 rather than in an exploratory state32. Acclimation periods of three or more weeks may reduce the number of pigs that pace in an experiment. However, if pacing persists throughout all the sampling periods, this ethogram may need to be adjusted to include walks and standing still.
Validity is the extent to which a measurement represents the intended scope of the question being asked25. When first developing the HAT, we used only a spatial ethogram. Definitions of spatial ethogram behavior describe accurately and specifically the proximity to the human subjects and they tell the observer directly how much space the pig leaves between itself and a human. However, once these methods are needed to be applied to a new laboratory set-up, we recognized that spatial ethograms are laboratory specific. Pen dimensions and the placement of other objects influence the outcome of the spatial ethogram; therefore, a diagram with measurements and specifics of the pen will need to be published if the pen set-up has not been previously reported. In addition to reporting on the pen environment, the structural behaviors were added to the ethogram. Unlike spatial behaviors, structural behaviors can be evaluated more easily across laboratories; these behaviors have validity because they specifically describe the pig&#39;s level of active state. When a pig is resting, it is likely not motivated to approach and is unable to change positions to approach as quickly as a standing pig. Similarly, a pig displaying NNOB is in an exploratory state, but a pig with its head still while standing is more likely in a catatonic state. The nose orientation helps with validity because the nose, ears, and then eyes are what the pig uses to gather information about the human.
Limitations of the Technique:
A potential concern with this technique is the variability in the pigs&#39; responses to the test-human.&#160;In addition, pigs will look at the test-human&#39;s hands, which can cause unintentional cueing by that person. Therefore, these limitations were expressed through the experimentally testing of 1) the pigs&#39; responses to a familiar human and unfamiliar humans, and 2) standardizing that, after the pellet is dropped, the test-human stands still and places their hands out of the pig&#39;s sight. Data showed that there were no treatment or treatment x time differences during the HAT (Table 2), suggesting that the HAT could be administered by either familiar or unfamiliar humans. Other researchers suggest that pigs tend to generalize about humans based on previous interactions11,12,13; therefore, a pig&#39;s previous experiences with humans need to be positive.&#160;This challenge can also be remedied with a vigilant experimental design; for each block, a sufficient number of experimental units represented for each treatment of interest is needed.
In this study, although there were only two experienced observers timestamping all the videos for all three housing types, there were differences among the housing types for specific behavior outcomes (Table 1). For example, pigs in housing type B entered the closest area more often than those in housing types A and C. This is likely due to a difference in the pen material; in housing type B, the front of the pen was a chain-linked gate with horizontal bars that allowed the pig to climb the gate during the HAT. Housing types A and C, on the other hand, had vertical bars and fewer horizontal surfaces for the pigs to climb on. This variability can be remedied by adding the duration spend in the close and closest areas before comparing them across housing types (Table 1; P &#62; 0.10). However, pigs in housing type C spent more time in the far area than those in housing types A and B (Table 1; P &#60; 0.05), which was likely due to the placement of waterers in the back of the pen rather than at the front of the pen. This is a limitation that can be remedied if laboratories choose to standardize the placement of waterers, bowls, and toys and make sure that they are fixed so that the pig does not move the object into another area.
This test has great accessibility for laboratories of all types, but, as mentioned previously, the manually stamped spatial ethogram and measurements will vary more across laboratories. Nonetheless, the body- and head-structural ethograms are ubiquitous. Laboratories that have access to validated, automated tracking for pigs may benefit by having the spatial ethogram tracked automatically rather than manually because the distance moved and the rate of movement may be additional outcomes of behavior measures from the HAT. The limitations from the in-pen set-up and traditional technologies rather than test areas and automatic tracking technologies may be remedied by adapting the AI formula. The AI provides standardized measurements and terminology for how individual pigs use their pen space and express interest in a human. This calculation, derived from common behavior measures, is sensitive to porcine models of subconcussive mTBI and, possibly, other states of subclinical injury or sickness. In addition, the AI reduces random variations during experimentation and may be more easily compared across experiments and laboratories than methods that rely on more experiment-specific measurements. The structural behaviors provided the foundation for this formula, because these behaviors are standard measurements across treatments, whereas the spatial behaviors are dependent on the pen set-up, number of pigs in a pen, and tracking system. For example, we observed that when two healthy pigs are tested in a pen, they will perform similar spatial behaviors by approaching together, but the pig that follows the first may orient its nose more towards its pen mate than towards the human and express more NNOB, because the pig who leads serves as the sentinel. Nonetheless, the AI aids in reducing this variation even from paired behaviors.
Although the AI is an excellent toolset for standardizing the test across laboratories, researchers may still want to examine test-specific behavior outcomes within a laboratory or experiment, especially if they have enough power (i.e., experimental units and repeated tests) in a single experiment. Therefore, Table 1, containing all behavior outcomes, the variance, the distribution, and a calculated test for the number of animals for each specific behavior, was included here. For example, if researchers have pen environments that allow pigs to consistently climb during the HAT and they know their treatment causes over a 75% difference in climbing behaviors, then they can justify animal numbers based on the variance measured. If new behaviors are added to the ethogram, scientists will need to justify which behaviors are indicative of approach or withdrawal before incorporating them into the index. For example, if most of the animals within an experiment pace along the walls of the pen (i.e., thigmotaxis)32, the duration of this behavior could be incorporated into the body-structural ethogram category. The behavior could be represented in the detailed stacked bar graph (i.e., Figure 5) or tabular form, and then, it can be summed up with stand-still before applying the index calculation. The AI, therefore, can represent behaviors that are ubiquitous across laboratories, but additional unique behaviors can still be represented separately.
Significance with Respect to Existing Methods:
The existing methods for the HAT were established for pigs on commercial farms to assess animal welfare. Here, a protocol for laboratory pigs has been established, which can help researchers to assess animal welfare and distinguish mTBI pigs from sham-treated pigs. An alternative traditional test could be to use an open-field test. This test was previously used to assess pig emotionality and welfare33. Open-field tests were originally designed for testing the affective states of rodents by measuring their natural aversion to open space and light. In contrast, healthy pigs may view the same stimuli as appetitive10, and after a sickness, injury, or stress treatment, they likely express fear. This test requires more laboratory space and will require pigs to acclimate to being handled and placed in an open-field arena. If laboratories have space and protocols for handling the pigs are in place, repeated HAT sessions, in addition to one open-field test, may help further distinguish treated animals from sham-treated animals.
Critical Steps Within the Protocol:
The first three steps in the protocol are the most critical for successful HAT measures. The sessions per pig only take 3 min; however, adequate preparation will help make this test reliable. As stated above, the camera location and recording set-up is crucial for clarity and replication. Improper camera angles can limit the observer&#39;s vision, which will add errors to the measurements. Another often overlooked step is fixing the objects in the pen. The pig will move unfixed objects, and this may affect its motivation to approach the human. The set-up and managing system are important because pigs need to be acclimated to their environments before they can perform the test consistently. Pigs that are not well-acclimated to their home pens or routine or are experiencing stress will defecate in other areas rather than at the back of the pen34. The defecation area may affect their motivation to approach. From the camera view, the observer should be able to identify individual pigs; however, it is important that the marking scheme does not provided information about the animal&#39;s treatment, as this will bias the observer25.
Identifying pigs is very important for obtaining the correct behavioral data for the right pig, even when they are single-housed. Pigs are often moved for their treatments, and a marking reinsures the observer that they are watching the same pig after it has been removed and placed back in its pen. Pigs can be housed in pairs, as in housing type C, and therefore, it becomes very important to identify the pigs. Livestock marking paints and markers require daily application; therefore, this protocol requires the use of a medical grade tape and a smear of tag cement. The tape sticks best to pigs with longer hair. Pigs with short hair and dry skin will slough off the tape more often than pigs with longer hair.
Future Applications:
In summary, the noninvasive in-pen HAT test described here is sensitive enough to detect mild and temporal dependent changes in pigs after mTBI. Moreover, we have developed a weighted index called the AI to evaluate changes in pigs housed in different pen types, as well as in different types of pig. Although the HAT has been used to detect changes in pigs exposed to mTBI, this behavioral test can be useful for detecting measurable behavioral changes in animals experiencing stress or prepathological conditions.

Human mTBI is often defined by functional deficits despite the absence of global structural changes or significant edema within the brain1,2,3. Indeed, in some mTBI patients, the characteristic feature of this injury is a change in their psychological state in the absence of any neuroanatomical changes4,5. We utilized a porcine model of mTBI6 because pig&#39;s brains are both anatomically and physiologically closer to humans than rodents7, and corresponding measurements could provide a relevant set of common data elements with humans.
In recent years, the porcine model has gained the interest of neurotrauma scientists and mTBI stakeholders for preclinical investigations; however, unlike rodent models of TBI, there are only a few standardized behavioral tests published that allow for the assessment of the laboratory pig&#39;s affective state (i.e., psychological state)7,8,9,10. A long-term goal for our laboratory is to develop several, complementary behavioral toolsets that are sensitive enough to measure when the pigs are experiencing subclinical sickness or when animals are in a prepathological stress-related state.
Repeated behavioral tests that measure the change of affective state in a laboratory pig may be good candidates for distinguishing an animal with a prepathological condition from healthy animals. For example, in-pen HATs were used for the commercial pig production to help farmers select healthy pigs with good temperament or modify management and housing strategies that caused distress, injury, and sickness11,12. These tests were used to quantify the motivation and overall affective state of one pig or a group of pigs13.
Our laboratory and other researchers measured motivation in pigs by quantifying three categories of behaviors: 1) exploratory states that are expressed through nonnutritive oral behaviors (NNOB), where the pig uses its mouth, snout, or face to sniff, lick, chew, and root a substrate, or they chomp without a substrate14,15; 2) spatial relationships of the pig to an object or being16; 3) nose direction, which is used instead of eye contact because pigs have monocular17, but near-sighted vision, and they prioritize their sense of smell over vision18. If a healthy pig associates humans with rewarding stimuli, they express a high frequency of NNOB, direct their nose towards the human, and seek to gain closer proximity to the human11,16. However, after sickness, injury, or a distressful experience, motivation to seek even pleasurable stimuli is reduced, and thus, these measurable behaviors are likely reduced19. Swine behavior researchers noted that anhedonia, the lack of motivation to experience pleasurable stimuli, is recognizable and measurable in pigs within their home environments20. Thus, repeated HATs (before and after treatment) may serve as a sensitive measure to distinguish laboratory pigs treated with subconcussive mTBI from sham-treated (anesthesia-only) subjects. Anhedonia is one affective state that TBI patients may experience21. The HAT used here has the potential in helping to streamline the translation of behavioral findings from an animal model to clinical work. HATs can be administered daily over the course of an experiment, which may also help standardize laboratory pigs&#39; care for optimizing animal welfare and husbandry22.
Here, using the HAT, the behavioral differences resulting from mTBI in mini-swine are probed. Minimizing behavioral variability was achieved by the utilizing noninvasive measures of the HAT and allowing the pigs to acclimate to their home pens, routine management, and a daily treat. Traditionally, a test arena is used to measure behaviors (e.g., open-field test). An in-pen test can be helpful in laboratories that have limited space. Moving and handling pigs in a test arena can cause a stress response (distress or eustress) and potentially add to the variation of the responses to the test. An in-pen test removes that handling component, and therefore, likely reduces variation from handling-stress17. For these reasons stated above, we developed a daily, in-pen HAT for this mTBI model.
Standardized and quantified measures that appropriately define the affective state of the animal are important aspects in developing a new behavior test. In addition, tests should be repeatable across multiple laboratories. Here, for developing this protocol, the HAT was tested in three laboratories&#39; different housing systems. Three subethograms were created to timestamp specific behaviors from sample videos. Next, a weighted formula was created to incorporate the three ethograms and allow for the use of HATs across multiple laboratories. Although this test was developed and used specifically for mini-swine treated with subconcussive mTBI, the methods and protocol developed here will have applications for distinguishing the difference between a subclinically injured/sick or distressed pig and a healthy pig.
Behavior outcomes can be influenced by single vs. group housing, free-space allowance, the type of floor used, the type of fence used, the location of feeding and water, the defecation area, and the environmental enrichment location. Therefore, three housing types was examined (Figure 1): housing type A was at Kansas State University (Manhattan, KS); housing type B and C were at Virginia Tech University (Arlington, VA). The individual Institutional Animal Care and Use Committee (IACUC) at each location approved the use of the facilities and procedures.
For developing the ethogram of housing type A (Figure 1A), Minnesota-cross mini-swine (boars = 7, gilt = 1; National Swine Research Resource Center, Columbia, MO; age = 25.6 &#177; 3.66 [mean &#177; standard deviation (SD)] weeks) were housed indoors in single pens with animal-friendly flooring (IACUC #3881). The pigs used for this protocol were in good health did not have treatments applied. For developing the protocol for housing type B, Yucatan mini-pigs (age = 25.3 &#177; 2.80 weeks [mean &#177; SD] were single-housed (Figure 1B) at Virginia Tech facilities (IACUC #15-060). Animal treatments are described elsewhere29 and included the induction of subconcussive mTBI using blast-wave overpressure or sham controls (anesthesia only). For developing the protocol of housing type C, five female G&#246;ttingen mini-pigs (age = 23.7 &#177; 1.18 weeks [mean &#177;SD]) were pair-housed at Virginia Tech in a large pen (Figure 1C; IACUC #15-060). The first two housing environments are typical laboratory housing or contain single-housed pigs. Housing type C is an atypical housing environment that can house two or more pigs and may be considered more of an enriched environment than standard laboratory housing. This protocol can be used across housing types if the following methods are followed.

<table><tbody><tr><td>Dome 3.0 Megapixel Cameras with 2.8-12 mm lens set between 2.8-3.2 mm</td><td>Points North Surveillance, Auburn, ME</td><td>CDL7233S</td><td>Lower mm lenses are needed for low-profile pens</td></tr><tr><td>Manfrotto 244 friction arm kit</td><td>B&amp;amp;H Photo</td><td>B&amp;amp;H # MA244; MFR # 244</td><td>To mount and secure cameras at a 90&amp;deg; angle</td></tr><tr><td>Video Recording System</td><td>Points North Surveillance, Auburn, ME</td><td>NVR-RACK64</td><td>NVR is customized</td></tr><tr><td>Colored and patterned duct tape attached to a double-sided medical grade tape</td><td>&amp;nbsp;MBK Tape Solutions, Chatsworth, CA</td><td>3M 1522H</td><td>Sustainable marking of pigs</td></tr><tr><td>Approach Index Formula generator</td><td>Dinasym, Manhattan, KS</td><td>Approach Formula</td><td>Company will customize macros for specific lab needs</td></tr><tr><td>Geovision Software</td><td>Points North Surveillance, Auburn, ME</td><td>Geovision</td><td>Software to edit video time into 180 s clips</td></tr><tr><td>Clicker</td><td>Petco</td><td>Good2Go Dog Training Clicker</td><td></td></tr><tr><td>Reward treat (feed pellet, carob chip, raisin, marshmallow)</td><td>Variable</td><td>N/A</td><td>Depending on previous exposure, adult pigs are very&amp;nbsp; neophobic when new food is introduced. Limit-fed pigs can be fed a few pellets of feed.&amp;nbsp;</td></tr><tr><td>Statistical Analysis System (SAS)</td><td>SAS Institute, Cary, North Carolina</td><td>SAS 9.0</td><td>Our laboratories preference for analyzing mixed models and repeated measures</td></tr><tr><td>Observer 11.5 software</td><td>Noldus Information Technology, Leesburg, VA</td><td>Observer 11.5</td><td>Software to manually timestamp video clips</td></tr></tbody></table>

noninvasive, in-pen approach test for laboratory-housed pigs

Traumatic brain injury (TBI) incidences have increased in both civilian and military populations, and many researchers are adopting a porcine model for TBI. Unlike rodent models for TBI, there are few behavioral tests that have been standardized. A larger animal requires more invasive handling in test areas than rodents, which potentially adds stress and variation to the animals&#39; responses. Here, the human approach test (HAT) is described, which was developed to be performed in front of laboratory pigs&#39; home pen. It is noninvasive, but flexible enough that it allows for differences in housing set-ups.
During the HAT, three behavioral ethograms were developed and then a formula was applied to create an approach index (AI). Results indicate that the HAT and its index, AI, are sensitive enough to detect mild and temporary alterations in pigs&#39; behavior after a mild TBI (mTBI). In addition, although specific behavior outcomes are housing-dependent, the use of an AI reduces variation and allows for consistent measurements across laboratories. This test is reliable and valid; HAT can be used across many laboratories and for various types of porcine models of injury, sickness, and distress. This test was developed for an optimized manual timestamping method such that the observer consistently spends no more than 9 min on each sample.

Watch this Scientific Journal Video about Noninvasive, In-pen Approach Test for Laboratory-housed Pigs at JoVE.com

Noninvasive, In-pen Approach Test for Laboratory-housed Pigs

This protocol describes a new behavioral test&#8212;the human approach test in the pigs&#39; home pen&#8212;to detect functional deficits in laboratory pigs after subconcussive traumatic brain injury.

Traumatic brain injury (TBI) incidences have increased in both civilian and military populations, and many researchers are adopting a porcine model for ...

noninvasive-in-pen-approach-test-for-laboratory-housed-pigs

Research

JoVE Journal

Behavior

8.3K Views.  Kansas State University. Deze methode kan helpen bij het beantwoorden van belangrijke vragen op het gebied van neurowetenschappen, met name over neurotrauma. Het belangrijkste voordeel van deze techniek is dat het geen arena of extra behandeling van het onderwerp vereist. Mijn onderzoeksteam kan worden gezien het aantonen van procedures in het protocol. Dit omvat Eduarda Bortoluzzi, Mikayla Goering, Luke Ruiz, Jared Mumm, en Morgan Coffin.Before het plaatsen van de dieren in hun hokken vast te stellen camera's op een...

Video: Noninvasive, In-pen Approach Test for Laboratory-housed Pigs

Basic Surgical Techniques in the G&ouml;ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion

The emergence of the G&ouml;ttingen minipig in research of topics such as neuroscience, toxicology, diabetes, obesity, and experimental surgery reflects the close resemblance of these animals to human anatomy and physiology 1-6.The size of the G&ouml;ttingen minipig permits the use of surgical equipment and advanced imaging modalities similar to those used in humans 6-8. The aim of this instructional video is to increase the awareness on the value of minipigs in biomedical research, by demonstrating how to perform tracheal intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion. 
Endotracheal Intubation should be performed whenever a minipig undergoes general anesthesia, because it maintains a patent airway, permits assisted ventilation and protects the airways from aspirates. Transurethral bladder catheterization can provide useful information about about hydration state as well as renal and cardiovascular function during long surgical procedures. Furthermore, urinary catheterization can prevent contamination of delicate medico-technical equipment and painful bladder extension which may harm the animal and unnecessarily influence the experiment due to increased vagal tone and altered physiological parameters. Arterial and venous catheterization is useful for obtaining repeated blood samples and monitoring various physiological parameters. Catheterization of femoral vessels is preferable to catheterization of the neck vessels for ease of access, when performing experiments involving frame-based stereotaxic neurosurgery and brain imaging. When performing vessel catheterization in survival studies, strict aseptic technique must be employed to avoid infections6. Transcardial perfusion is the most effective fixation method, and yields preeminent results when preparing minipig organs for histology and histochemistry2,9. For more information about anesthesia, surgery and experimental techniques in swine in general we refer to Swindle 2007. Supplementary information about premedication and induction of anesthesia, assisted ventilation, analgesia, pre- and postoperative care of G&ouml;ttingen minipigs are available via the internet at <a href="http://www.minipigs.com">http://www.minipigs.com</a>10. For extensive information about porcine anatomy we refer to Nickel et al. Vol. 1-511.

Basic Surgical Techniques in the G&#246;ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion

The use of domestic and miniature pigs in science has increased significantly in recent years. By demonstrating how to perform intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion, we aim to further increase the value of G&ouml;ttingen minipigs in biomedical research.

The emergence of the G&ouml;ttingen minipig in research of topics such as neuroscience, toxicology, diabetes, obesity, and experimental surgery reflects ...

Medicine

Transition of Farm Pigs to Research Pigs using a Designated Checklist followed by Initiation of Clicker Training - a Refinement Initiative

Implementation of 3R initiatives is important. To refine the life of laboratory pigs, we focused on the time period prior to inclusion of animals in experiments. We improved the checklist during the development. By using the checklist each individual pig&#39;s progress was documented. This aids the caretakers so they remain focused on the actions necessary to socialize the animals and limit the animal&#39;s stress and anxiety level. During this phase, the pigs become calm and ready to commence additional clicker training to further limit stress during the experimental period. The pigs will readily allow basic outcome measures to be collected without sedation or fixation.
The protocol is divided in two. First the transition phase that begins as the pigs arrive at the research facility. Introducing treats and gradually closer contact to the caretakers are the first steps. Then, the caretaker introduces actions to be able to touch the head and hind, to mimic e.g. manipulating the ear to take a blood sample or the hind to obtain a rectal temperature. A strong foundation is established in the animal-caretaker relationship and this swiftly minimizes the stress the pigs express after arrival. Secondly, the positive reinforcement training commences to teach the animal how to carry out more complex tasks without becoming stressed. A clicker is introduced as a secondary reinforcer.
Time, structure, education and thorough communication seem to be the most important criteria to succeed in the application of this protocol. In addition to having calm and compliant pigs, the caretakers report that they enjoy the focus they are allowed to give each individual pig, adding to their overall job satisfaction. Having caretakers with a strong commitment, and a focus on the transition progress and welfare of each pig, additionally strengthens the department&#39;s Culture of Care.

Refinement of porcine studies is achieved by introducing a standardized checklist and positive reinforcement training using a clicker. This work supports the collection of samples and the conduct of daily chores related to the animals.

Implementation of 3R initiatives is important. To refine the life of laboratory pigs, we focused on the time period prior to inclusion of animals in ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Immunology and Infection

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder regions, collectively referred to as the brachial plexus (BP). Despite recent advances in obstetrical care, the problem of NBPP continues to be a global health burden with an incidence of 1.5 cases per 1,000 live births. More severe types of this injury can cause permanent paralysis of the arm from the shoulder down. Prevention and treatment of NBPP warrants an understanding of the biomechanical and physiological responses of newborn BP nerves when subjected to stretch. Current knowledge of the newborn BP is extrapolated from adult animal or cadaveric BP tissue instead of in vivo neonatal BP tissue. This study describes an in vivo mechanical testing device and procedure to conduct in vivo biomechanical testing in neonatal piglets. The device consists of a clamp, actuator, load cell, and camera system that apply and monitor in vivo strains and loads until failure. The camera system also allows monitoring of the failure location during rupture. Overall, the presented method allows for a detailed biomechanical characterization of neonatal BP when subjected to stretch.

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Presented here are methods to perform in vivo biomechanical testing on brachial plexus in a neonatal piglet model.

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder ...

Bioengineering

Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question the safety of anesthesia in children, implicating anesthetics as potentially toxic to the brain in development. To date, no studies have successfully elucidated the mechanism(s) by which anesthesia may be neurotoxic. Animal studies allow investigation of such mechanisms, and neonatal piglets represent an excellent model to study these effects due to their striking developmental similarities to the human brain.
This protocol adapts the use of enzyme-based microelectrode array (MEA) technology as a novel way to study the mechanism(s) of anesthesia-induced neurotoxicity (AIN). MEAs enable real-time monitoring of in vivo neurotransmitter activity and offer exceptional temporal and spatial resolution. It is hypothesized that anesthetic neurotoxicity is caused in part by glutamate dysregulation and MEAs offer a method to measure glutamate. The novel implementation of MEA technology in a piglet model presents a unique opportunity for the study of AIN.

This study explores the novel use of enzyme-based microelectrode array (MEA) technology to monitor in vivo neurotransmitter activity in piglets. The hypothesis was that glutamate dysregulation contributes to the mechanism of anesthetic neurotoxicity. Here, we present a protocol to adapt MEA technology to study the mechanism of anesthesia-induced neurotoxicity.

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question ...

Neuroscience

Non-Terminal Blood Sampling Techniques in Guinea Pigs

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently represents a relatively narrow area of research and descriptive data on specific methodology is correspondingly scarce. The anatomical features of guinea pigs are slightly different from other rodent models, hence modulation of sampling techniques to accommodate for species-specific differences, e.g., compared to mice and rats, are necessary to obtain sufficient and high quality samples. As both long and short term in vivo studies often require repeated blood sampling the choice of technique should be well considered in order to reduce stress and discomfort in the animals but also to ensure survival as well as compliance with requirements of sample size and accessibility. Venous blood samples can be obtained at a number of sites in guinea pigs e.g., the saphenous and jugular veins, each technique containing both advantages and disadvantages4,5. Here, we present four different blood sampling techniques for either conscious or anaesthetized guinea pigs. The procedures are all non-terminal procedures provided that sample volumes and number of samples do not exceed guidelines for blood collection in laboratory animals6. All the described methods have been thoroughly tested and applied for repeated in vivo blood sampling in studies within our research facility.

Though a known model, the guinea pig currently represents a niche in experimental animal sciences and limited data is available on the execution of most procedures. Here we present four different approaches to non-terminal in vivo blood sampling techniques in either conscious or anaesthetized guinea pigs.

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently ...

Biology

Endotracheal Intubation Using a Flexible Intubation Endoscope as a Standardized Model for Safe Airway Management in Swine

Endotracheal intubation is often a basic requirement for translational research in porcine models for various interventions that require a secured airway or high ventilation pressures. Endotracheal intubation is a challenging skill,&#160;requiring a minimum number of successful endotracheal intubations to achieve a high success rate under optimal conditions, which is often unachievable for non-anaesthesiology researchers. Due to the specific porcine airway anatomy, a difficult airway can usually be assumed. The impossibility of establishing a secure airway can result in injury, adverse events, or death of the laboratory animal. Using a prospective, randomized, controlled evaluation approach, it has been shown that fiberoptic-assisted endotracheal intubation takes longer but has a higher first-pass success rate than conventional intubation without causing clinically relevant drops in oxygen saturation. This model presents a standardized regimen for endoscopically guided endotracheal intubation, providing a secured airway, especially for researchers who are inexperienced in the technique of endotracheal intubation via direct laryngoscopy. This procedure is expected to minimize animal suffering and unnecessary animal losses.

The use of pigs in research has increased in recent years. Nevertheless, pigs are characterized by difficult airway anatomy. By demonstrating how to perform endoscopically guided endotracheal intubation, the present protocol aims to further increase laboratory animals' safety to avoid animal suffering and unnecessary death.

Endotracheal intubation is often a basic requirement for translational research in porcine models for various interventions that require a secured airway ...

Transdermal Measurement of Glomerular Filtration Rate in Mechanically Ventilated Piglets

Transdermal measurement of glomerular filtration rate (GFR) has been used to evaluate kidney function in conscious animals. This technique is well established in rodents to study acute kidney injury and chronic kidney disease. However, GFR measurement using the transdermal system has not been validated in pigs, a species with a similar renal system to humans. Hence, we investigated the effect of sepsis on transdermal GFR in anesthetized and mechanically ventilated neonatal pigs. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP). The transdermal GFR measurement system consisting of a miniaturized fluorescence sensor was attached to the pig's shaved skin to determine the clearance of fluorescein-isothiocyanate (FITC) conjugated sinistrin, an intravenously injected GFR tracer. Our results show that at 12 h post-CLP, serum creatinine increased with a decrease in GFR. This study demonstrates, for the first time, the utility of the transdermal GFR approach in determining renal function in mechanically ventilated, neonatal pigs.

Glomerular filtration rate (GFR) is the ideal marker for assessing kidney function. However, the standard measurement method using inulin injection with serial blood and urine analysis is impractical. This article delineates a practical method to measure GFR transdermally in piglets.

Transdermal measurement of glomerular filtration rate (GFR) has been used to evaluate kidney function in conscious animals. This technique is well ...

Echocardiography Recording in Awake Miniature Pigs

Echocardiography uses ultrasonic waves to non-invasively assess cardiac structure and function and is the standard of care for cardiac assessment and monitoring. The miniature pig, or minipig, is increasingly being used as a model of cardiac disease in medical research. Pigs are notoriously difficult to restrain and handle safely, and, therefore, research echocardiography in this species is almost always performed under anesthesia or heavy sedation. Anesthetics and sedatives universally affect cardiovascular function and may cause the depression of cardiac output and blood pressure, increases or decreases in heart rate and systemic vascular resistance, changes in the electrical rhythm, and altered coronary blood flow. Therefore, sedated or anesthetized echocardiography may not accurately depict the progression of cardiac disease in large animal models, thereby limiting the translational value of these important studies. This paper describes a novel device that allows for standing awake echocardiography in minipigs. In addition, training techniques used to teach pigs to tolerate this painless and non-invasive procedure without the need for hemodynamic-altering anesthetics are described. Standing awake echocardiography represents a safe and feasible way to perform the most common cardiac monitoring test in minipigs for cardiovascular research.

A simple cart construct, built to perform research echocardiography in standing awake minipigs, is described, along with building considerations, training techniques, and representative ultrasound images.

Echocardiography uses ultrasonic waves to non-invasively assess cardiac structure and function and is the standard of care for cardiac assessment and ...

Noninvasive, In-pen Approach Test for Laboratory-housed Pigs

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