Beatrice Tscherning Olesen, Karoline Jakobsen, Tine Bj&#248;rnholt Gustafsen, Helena C&#233;line Kj&#230;r Byriel and Kira Sonnichsen Graahede are acknowledged for their great efforts, feedback and assistance in designing the checklist and using it in their daily work. Medical illustrator Ken Kragsfeldt is acknowledged for his professional help illustrating figure 3.

The pigs used to validate the protocol were part of studies approved by The Animal Experimental Council - a national governmental institution evaluating and approving all animal experiments in Denmark. The Council does the ethical review similar to an IACUC.
1. Arrival at the research facility
<ol>
	<li>First, wash the pigs in a designated area to allow a thorough visual inspection. Use an exclusion list to exclude pigs with wounds, ear bites, abscesses, lameness etc., so only animals that appear clinically healthy are allowed into the facility.</li>
	<li>After washing, sort the pigs into groups according to color marks. The color marks indicate which pigs have been pen mates at the supplier. This is important since pigs will fight to establish a hierarchy when they are introduced to new pen mates. Keeping them sorted in groups together with pigs they are already familiar with limits this fighting considerably.</li>
	<li>Leave the pigs undisturbed (except when they are fed in the afternoon) for the rest of the arrival day as they can be quite stressed after transportation.</li>
</ol>
2. Part one: The transition phase (estimated time 4-6 days)
NOTE: The pigs stay in their home pen during the activities performed in the transition phase. We use half an apple for each treat-session. The progress of each individual pig is scored in the Transition Phase Progression Checklist (Figure 1 and Supplementary File 1).
<ol>
	<li>On the two consecutive days after arrival, introduce the treats.
	<ol>
		<li>Toss treats a few at a time (half apple total) into the pen twice daily to allow the pigs to search for the treats on their own and stand quietly just outside the pen. Apart from becoming familiar with treats, the goal is for the animal to positively associate the treat with the presence of a caretaker.</li>
	</ol>
	</li>
	<li>On day three and successive days introduce familiarizing the pig with &#34;handling by a caretaker&#34;. Enter the pen during feeding.
	<ol>
		<li>Sit down at arm&#39;s length from the pig while it is eating (Figure 3). Calmly extend a hand towards the pig to touch its back. The touch should be more than two seconds to count as successful. If the pig backs away from the food, withdraw the hand slowly.</li>
		<li>Wait until the animal commences eating and perform the action again. If the animal backs away from the food again, stop trying to touch it but remain inside the pen next to the trough (2-3 minutes in total for the session).</li>
	</ol>
	</li>
	<li>Move on in the handling procedure when the pig accepts being touched on the back while it is eating. Now move the hand around on the back in both direction of the head and the tail.
	<ol>
		<li>Touch the head and ears if the pig accepts it. If at any time, the pig backs away from an action stop the action but remain inside the pen for the remaining time (2-3 minutes in total for the session). The ear is important as it enables blood sampling from an intravenous catheter in the ear.</li>
		<li>Rise slowly to be able to touch the tail region which is the second important area. Standing and moving about is more frightening to the pig, which is why this step is scored independently. When the pig accepts having the area touched and the tail manipulated, a rectal temperature can be taken to support most research protocols.</li>
	</ol>
	</li>
	<li>To further strengthen the bond between caretaker and animal enter the pen during two sessions apart from the feeding times. Offer the pig treats (one slice at a time) during two sessions (2-3 minutes each), while sitting in a corner inside the pen. At first, give the treats from an outstretched arm, but decrease the distance gradually so the pig needs to come closer to get its reward.</li>
	<li>The final stage of the transition phase is being able to touch the pig in the specific areas of the body and using only a few treats to do so. Enter the pen while a second caretaker stands outside the pen and offers a treat or two to the pig. Reward the pig for being calm and allowing touching as described and at the same time being capable of keeping all four hoofs on the ground (not jumping up on the fence of the pen).</li>
</ol>
3. Part two: Commencing clicker training
NOTE: Following the transition phase, the animals are ready to start the clicker training. It is recommended that the training is performed in a designated area as it helps the pig to recognize that a training session is about to begin. Several short sessions across the day are better than one longer session.
<ol>
	<li>Take one pig at a time outside the home pen and into the designated training area. If the area used is novel to the pig, the first sessions should be without training and strictly exploratory with a few treats during about 5-7 minutes exploration per pig.</li>
	<li>Introduce the clicker sitting or standing with the treats out of reach of the pig. Click when the pig shows interest, and immediately give a treat. Do this in quick succession while the pig maintains interest. If the pig loses interest, pause the click-and-treat until the pig makes contact again. This session should be no longer than 10 minutes.</li>
	<li>The pig will understand the click quickly, but it is important to make the association between click and treat very strong. Repeat the step a few sessions in a row to make the association between click and treat strong. Test if the pig associates the click with a treat by clicking once and not following this with a treat and monitoring the behavior of the pig closely. If the pig looks around eagerly expecting the treat, the training is complete, if not perform a few additional sessions.</li>
	<li>Once the click is known by the pig, introduce the target stick. A target stick is a stick with a marked area on one end. Hold the target end in the height of the snout. At some point, the pig will touch it out of curiosity. Click in the very second that the pig touches the target and give a treat.</li>
	<li>Present the target again immediately and if touched click again and give a treat. Continue the target training until the pig understands that it will be rewarded for touching the target. Move the target gradually further away so the pig has to move further to get to the target.</li>
	<li>When the pig follows the target when it is moved around add additional challenges by putting objects into the training area, so the pig has to concentrate harder on the target stick task to get its rewards.</li>
</ol>

<ol>
	<li>Burch, R. L., Russell, W. M. 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> The principles of humane experimental technique. , (1959).</li><li>The 3Rs. National Centre for the Replacement Available from: <a target="_blank" href="https://www.nc3rs.org.uk/the-3rs">https://www.nc3rs.org.uk/the-3rs</a> (2021)</li><li>Gouveia, K., Hurst, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimising+reliability+of+mouse+performance+in+behavioural+testing:+the+major+role+of+non-aversive+handling.">Optimising reliability of mouse performance in behavioural testing: the major role of non-aversive handling.</a> Scientific Reports. 7, (2017).</li><li>Laule, G. E., Thurston, R. H., Alford, P. L., Bloomsmith, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Training+to+reliably+obtain+blood+and+urine+samples+from+a+diabetic+chimpanzee+(Pan+troglodytes).">Training to reliably obtain blood and urine samples from a diabetic chimpanzee (Pan troglodytes).</a> Zoo Biology. 15 (6), 587-591 (1996).</li><li>Mills, D., Ledger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+oral+selegiline+hydrochloride+on+learning+and+training+in+the+dog:+A+psychobiological+interpretation.">The effects of oral selegiline hydrochloride on learning and training in the dog: A psychobiological interpretation.</a> Progress in Neuro-Psychopharmacology &amp; Biological Psychiatry. 25 (8), 1597-1613 (2001).</li><li>Arluke, A., Sanders, C. 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> Regarding Animals. , (1996).</li><li>Leidinger, C., Herrmann, F., Thone-Reineke, C., Baumgart, N., Baumgart, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introducing+Clicker+Training+as+a+Cognitive+Enrichment+for+Laboratory+Mice.">Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice.</a> Journal of Visualized Experiments. (121), e55415 (2017).</li><li>Leidinger, C. S., Kaiser, N., Baumgart, N., Baumgart, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Clicker+Training+and+Social+Observation+to+Teach+Rats+to+Voluntarily+Change+Cages.">Using Clicker Training and Social Observation to Teach Rats to Voluntarily Change Cages.</a> Journal of Visualized Experiments. (140), e58511 (2018).</li><li>Sorensen, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Never+wrestle+with+a+pig.">Never wrestle with a pig.</a> Lab Animal. 44 (2), 159-161 (2010).</li><li>. 2019 report on the statistics on the use of animals for scientific purposes in the Member States of the European Union in 2015-2017 Available from: <a target="_blank" href="https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:52020DC0016&amp;from=EN">https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:52020DC0016&amp;from=EN</a> (2020)</li><li>Louise, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+training+pigs+within+an+isolation+unit+(a+pilot+study.">Target training pigs within an isolation unit (a pilot study.</a> Animal Technology and Welfare. 17 (1), 46-47 (2018).</li><li>Ryden, A., 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=Nursing+and+training+of+pigs+used+in+renal+transplantation+studies.">Nursing and training of pigs used in renal transplantation studies.</a> Laboratory Animals. , (2019).</li><li>Smith, A. C., Swindle, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+swine+for+the+laboratory.">Preparation of swine for the laboratory.</a> ILAR Journal. 47 (4), 358-363 (2006).</li><li>Swindle, M. M., Smith, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Swine in the Laboratory: Surgery, Anesthesia, Imaging, and Experimental Techniques, Third Edition. , (2016).</li><li>Obernier, J. A., Baldwin, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+an+appropriate+period+of+acclimatization+following+transportation+of+laboratory+animals.">Establishing an appropriate period of acclimatization following transportation of laboratory animals.</a> ILAR Journal. 47 (4), 364-369 (2006).</li><li>Kaiser, A. R., Johnson, A. K., Ross, J. W., Selsby, J. T., Stalder, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+Study+490A:+Positive+Reinforcement+Training+Piglets+to+Stand+in+a+Container+and+Follow+a+Human.">Independent Study 490A: Positive Reinforcement Training Piglets to Stand in a Container and Follow a Human.</a> Iowa State University Animal Industry Report. 11 (1), (2014).</li><li>S&#248;rensen, D. B., Dragsted, N., Glerup, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+reinforcement+training+in+large+experimental+animals.">Positive reinforcement training in large experimental animals.</a> Altex. 28, 250 (2011).</li><li>Lawrence, A. B., Vigors, B., Sandoe, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+Is+so+Positive+about+Positive+Animal+Welfare?-A+Critical+Review+of+the+Literature.">What Is so Positive about Positive Animal Welfare?-A Critical Review of the Literature.</a> Animals. 9 (10), (2019).</li><li>Jirkof, P., Rudeck, J., Lewejohann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+Affective+State+in+Laboratory+Rodents+to+Promote+Animal+Welfare-What+Is+the+Progress+in+Applied+Refinement+Research.">Assessing Affective State in Laboratory Rodents to Promote Animal Welfare-What Is the Progress in Applied Refinement Research.</a> Animals. 9 (12), (2019).</li><li>Herskin, M. S., Bundgaard, C. J., Ottesen, J. L., S&#248;rensen, D. B., Marchant-Forde, J. N., S&#248;rensen, D. B., Cloutier, S., Gaskill, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Animal-centric care and management. , 173-186 (2020).</li><li>What is a Culture of Care. Available from: <a target="_blank" href="https://norecopa.no/more-resources/culture-of-care">https://norecopa.no/more-resources/culture-of-care</a> (2020)</li><li>Bertelsen, T. H., S&#248;rensen, D. B., Cloutier, S., Gaskill, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+.+eds+Dorte+Bratbo+S&#248;rensen.">in . eds Dorte Bratbo S&#248;rensen.</a> Animal-centric care and management. , 15-29 (2020).</li><li>Weichbrod, R. H., Thompson, G. A., Norton, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Management of Animal Care and Use Programs in Research, Education, and Testing. 2 edn. , (2017).</li><li>SPF-DANARK. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPF+status+designations.">SPF status designations.</a> SPF-DANARK. , (2021).</li><li>Berset Convenor, F., 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=Federation+of+European+Laboratory+Animal+Science+Associations+recommendations+of+best+practices+for+the+health+management+of+ruminants+and+pigs+used+for+scientific+and+educational+purposes.">Federation of European Laboratory Animal Science Associations recommendations of best practices for the health management of ruminants and pigs used for scientific and educational purposes.</a> Lab Animal. , (2020).</li><li>Rehbinder, 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=FELASA+recommendations+for+the+health+monitoring+of+breeding+colonies+and+experimental+units+of+cats,+dogs+and+pigs.+Report+of+the+Federation+of+European+Laboratory+Animal+Science+Associations+(FELASA)+Working+Group+on+Animal+Health.">FELASA recommendations for the health monitoring of breeding colonies and experimental units of cats, dogs and pigs. Report of the Federation of European Laboratory Animal Science Associations (FELASA) Working Group on Animal Health.</a> Lab Animal. 32 (1), 1-17 (1998).</li></ol>

The authors have no disclosures.

The implementation of a protocol that includes part one, a transition phase checklist and part two, a consecutive clicker training program is a confirmed success. Introduction and use of a detailed checklist have provided a way to refine the introduction of pigs to the research facility. The structured transition phase furthermore creates a better foundation to train the pigs prior to inclusion in research projects. It has been a positive surprise to notice that the caretakers&#39; commitment to the work increases and th...

The principles of the 3Rs (Replacement, Reduction and Refinement) discussed by Russell and Burch in 1959, form today&#39;s foundation for high ethical standards concerning the use of animals for experimental purposes. In biomedical research facilities engaged in preclinical laboratory animal studies, great importance must be adhered to the refinement principle. &#34;Refinement means any decrease in the incidence or severity of inhumane procedures applied to those animals which still have to be used&#34;1.
A contemporary definition additionally states that refinement is &#34;advancing research into animal welfare by exploiting the latest in vivo technologies and by improving understanding of the impact of welfare on scientific outcomes&#34;2. This implies that not only are today&#39;s refinement initiatives important for the welfare of the animal per se, but that they are also important to research quality as scientific outcomes will benefit from these initiatives 3.
One refinement initiative to consider is to socialize and train experimental animals. They may be trained to perform a certain task e.g., step onto a scale or behave calmly while blood samples are drawn. Some species may be more naturally disposed to training than others and this impacts the tradition for training laboratory animal species. Traditionally, training of dogs has been performed for centuries e.g., in order to use the dog in hunting. These historic traditions have most certainly made the training of dogs in the biomedical research setting more straightforward. Together with non-human primates, training of laboratory dogs has been discussed and undertaken for quite some time4,5.
Common to both of these species is that they rank high on the &#34;socio-zoological scale&#34;. This scale ranks animals according to their place and use in human society 6. Ranking high also carries with it a large public demand to treat the animals as humanely as possible and limit their stress and anxiety level. Rodents, in contrast to dogs and non-human primates, are placed in the lower end of the socio-zoological scale. They have not received equivalent public attention and care. Historically they have been treated less well - presumably also due to their ability to spread life-threatening disease. However, during the last few years the training of rodents has been implemented in preclinical research settings and hereof related scientific benefits have not gone unnoticed. Both rats and mice are interestingly quite easily trained and the training also limits their stress-response and thus strengthens scientific outcomes7,8.
Reporting on how to socialize and train pigs in a laboratory animal setting is to our knowledge scarce9. On the socio-zoological scale, pigs are placed in-between dogs and rodents. We recognize the species primarily as a food resource, but nevertheless quite a large number of pigs are also used in biomedical research. In the EU alone, approximately 75,000 pigs were used annually for scientific purposes in 2015-2017 10. This number encompasses both farm pigs and miniature pigs. There is an increasing focus on refining the lives of these animals by training as a measure to comply with the 3Rs. Both farm pigs and miniature pigs can indeed be trained 11,12, but their starting point in terms of socialization varies. Miniature pigs are bred for research and socialized during their early life span. They are bred to have a calm temper. Farm pigs are bred for consumption and often not socialized at all. Finisher pigs for meat production in Denmark are typically housed in high density groups of 15-25 pigs where they have to compete to earn a position in the herd&#39;s hierarchy. Their temper reflects that ability. Often, they are housed in stable facilities with a partially slated floor to limit the workload of mucking out. To optimize weight gain, they are fed ad libitum through automated food and water delivering systems. Hence, their contact with farm workers is limited and they will become stressed when handled. This poses certain challenges when the animals are transferred to a research facility. Here the animal welfare, the husbandry, and the 3Rs play a key role13,14. From a research perspective, each individual animal is important to obtain&#160;the best possible research results. The welfare of the animals is also important to ensure a low inter-animal variation in regard to stress, anxiety and fear. For these reasons it is important to pay attention to the transition from a &#34;herd animal&#34; to an &#34;individual&#34;. Upon arrival, the pigs enter a new environment with new scents, new food, day-to-day contact with animal caretakers and often also new pen mates. To a pig, the shift can be very stressful and the importance of an acclimatization period of several days is acknowledged in laboratory animal science15.
By structuring the acclimatization period, we have created what we call a &#34;transition phase&#34; using a structured step-by-step checklist. The checklist ensures that each pig receives sufficient caretaker contact and learns to allow basic handling for examination and sample collection without being stressed. Continuous work can then be carried out to train the pigs using positive reinforcement training targeted to each specific research project16,17. A clicker is used to signal a food reward when the desired action is performed. Combining the steps from the transition phase with additional clicker training of the pigs gives the animals a smooth settling in at the research facility. They become calm and easy to handle and can, with little effort, be taught to stand still for e.g. blood sample collection. By the use of this type of handling protocol, old-fashioned techniques such as sedation of the animals or snares are replaced. To make sure all pigs are handled sufficiently a checklist is filled out.
It is becoming widely accepted that animals do indeed feel positive emotions and so focus when working with the animals should not solely be on the removal of negative emotions 18. The training session with the caretaker is an example of a positive emotional experience. It is quite enjoyable for the pig as it receives treats and attention. Besides the benefit of reducing handling-stress and fear related behaviors at the facility, training the animals provides an opportunity to give the animals positive emotional experiences. The adding of positive experiences may counteract some of the negative experiences that laboratory animals encounter in relation to experiments performed and will thus further increase animal welfare 19,20.
While the pigs evidently enjoy the training sessions, the sessions simultaneously offer the caretaker an opportunity to strengthen the bond to each individual pig. Being an animal caretaker, performing painful actions, such as injections of the animals, is part of the job. However, being able to train the animals and turn these actions into positive experiences is rewarding in itself. It increases the commitment of the caretakers and plays a key role in strengthening the department&#39;s culture of care21,22,23.
The pigs were housed according to the Institution&#39;s Animal Welfare Policy, which refers to national legislation on animal experimentation, housing and husbandry. The protocol can be applied to weaned pigs of any age. For the development of the transition checklist (Figure 1) a total of 40 female crossbred farm pigs (Danish Landrace-Yorkshire-Duroc) weighing 35-80 kg were used (Table 1). The pigs were allocated to a group dependent on their arrival time. The pigs were housed in the research stable facility in the period January - September 2020. All farm pigs are bred on a traditional sow farm and moved to a collaborating farm when weighing approximately 15 kg. At least two weeks before inclusion in survival studies, the pigs are transported to the university&#39;s research stable facility. During the first two weeks the transition from farm pig to research animal takes place. The protocol is composed of non-harmful and positive actions and hence may be initiated immediately as part of the acclimatization period.
The pigs were housed one by one in pens measuring 3.4 - 6.8 m2 on solid concrete floors and have access to water via an automatic water supply. There is a feeding trough for each pig and at least one section of the separation to the neighboring pens allows snout contact. When pigs are scheduled to receive surgical interventions or have equipment implanted, they are housed alone to avoid pen mates licking and biting in wounds and pulling implants out. The bedding material is straw and the animals are further enriched with hay and different activity toys e.g. rope, balls, buckets, chew sticks made from plastic (Figure 2).
The animals come from a herd included in the national health-monitoring program for farm pigs, which means they are screened for seven pathogens24 that can affect pigs in a production setting. Biannual random checks are performed for the research herd based on FELASA recommendations25,26, in addition to samples from pigs that warrant diagnostics due to unexpected research results. Some of the pigs used for this project were seropositive for porcine reproductive and respiratory syndrome; however, none of the pigs showed any clinical symptoms corresponding to infection. All samples evaluated using polymerase chain reaction were negative, corroborating that the pigs were in a healthy condition.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Wrap, e-power, bl&#229;, 7,5cm</td>
			<td>E-vet A/S, Ole R&#248;mers Vej 26 A, DK-6100 Haderslev</td>
			<td>203165B</td>
			<td>Adhesive wrap used to wrap around the end of a stick to make a target stick. It is an advantage that it can be changed as it will get dirty quite quickly.</td>
		</tr>
		<tr>
			<td>Apples</td>
			<td>Varius</td>
			<td>Uknown</td>
			<td>Used as treats</td>
		</tr>
		<tr>
			<td>Klikker (Clicker)</td>
			<td>E-vet A/S, Ole R&#248;mers Vej 26 A, DK-6100 Haderslev</td>
			<td>TX2289</td>
			<td>Clicker used in training</td>
		</tr>
	</tbody>
</table>

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.

Applying the transition phase as described in the protocol&#39;s part one allows for a smooth introduction of farm pigs to the research facility. Having calm pigs to work with eases daily chores and enables minor procedures and sample taking to be performed without anesthesia or fixation.
To follow the progress of pigs during the transition phase a checklist (Checklist 1) was developed to mark when individual pigs reach defined steps in their transition. The pigs are scheduled to enter researc...

Watch this Scientific Journal Video about Transition of Farm Pigs to Research Pigs using a Designated Checklist followed by Initiation of Clicker Training - a Refinement Initiative at JoVE.com

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

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 ...

transition-farm-pigs-to-research-pigs-using-designated-checklist

Research

JoVE Journal

Behavior

5.1K Views.  Aarhus University. 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. 

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

Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous research determined a profound impact of gentle handling protocols on the well-being of laboratory mice. By introducing clicker training to the keeping of mice, not only do we promote the amicable treatment of mice, but we also enable them to experience cognitive enrichment. Clicker training is a form of positive reinforcement training using a conditioned secondary reinforcer, the "click" sound of a clicker, which serves as a time bridge between the strengthened behavior and an upcoming reward. The effective implementation of the clicker training protocol with a cohort of 12 BALB/c inbred mice of each sex proved to be uncomplicated. The mice learned rather quickly when challenged with tasks of the clicker training protocol, and almost all trained mice overcame the challenges they were given (100% of female mice and 83% of male mice). This study has identified that clicker training for mice strongly correlates with reduced fear in the mice during human-mice interactions, as shown by reduced anxiety-related behaviors (e.g., defecation, vocalization, and urination) and fewer depression-like behaviors (e.g., floating). By developing a reliable protocol that can be easily integrated into the daily routine of the keeping of laboratory mice, the lifetime experience of welfare in the mice can be improved substantially.

The development of new refinement strategies for laboratory mice is a challenging task that contributes towards fulfilling the 3R principle. This protocol introduces clicker training as a cognitive enrichment program for laboratory mice.

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous ...

Using a Split-belt Treadmill to Evaluate Generalization of Human Locomotor Adaptation

Understanding the mechanisms underlying locomotor learning helps researchers and clinicians optimize gait retraining as part of motor rehabilitation. However, studying human locomotor learning can be challenging. During infancy and childhood, the neuromuscular system is quite immature, and it is unlikely that locomotor learning during early stages of development is governed by the same mechanisms as in adulthood. By the time humans reach maturity, they are so proficient at walking that it is difficult to come up with a sufficiently novel task to study de novo locomotor learning. The split-belt treadmill, which has two belts that can drive each leg at a different speed, enables the study of both short- (i.e., immediate) and long-term (i.e., over minutes-days; a form of motor learning) gait modifications in response to a novel change in the walking environment. Individuals can easily be screened for previous exposure to the split-belt treadmill, thus ensuring that all experimental participants have no (or equivalent) prior experience. This paper describes a typical split-belt treadmill adaptation protocol that incorporates testing methods to quantify locomotor learning and generalization of this learning to other walking contexts. A discussion of important considerations for designing split-belt treadmill experiments follows, including factors like treadmill belt speeds, rest breaks, and distractors. Additionally, potential but understudied confounding variables (e.g., arm movements, prior experience) are considered in the discussion.

We describe a protocol for investigating human locomotor adaptation using the split-belt treadmill, which has two belts that can drive each leg at a different speed. We specifically focus on a paradigm designed to test the generalization of adapted locomotor patterns to different walking contexts (e.g., gait speeds, walking environments).

Understanding the mechanisms underlying locomotor learning helps researchers and clinicians optimize gait retraining as part of motor rehabilitation. ...

Using a Virtual Store As a Research Tool to Investigate Consumer In-store Behavior

People&#39;s responses to products and/or choice environments are crucial to understanding in-store consumer behaviors. Currently, there are various approaches (e.g., surveys or laboratory settings) to study in-store behaviors, but the external validity of these is limited by their poor capability to resemble realistic choice environments. In addition, building a real store to meet experimental conditions while controlling for undesirable effects is costly and highly difficult. A virtual store developed by virtual reality techniques potentially transcends these limitations by offering the simulation of a 3D virtual store environment in a realistic, flexible, and cost-efficient way. In particular, a virtual store interactively allows consumers (participants) to experience and interact with objects in a tightly controlled yet realistic setting. This paper presents the key elements of using a desktop virtual store to study in-store consumer behavior. Descriptions of the protocol steps to: 1) build the experimental store, 2) prepare the data management program, 3) run the virtual store experiment, and 4) organize and export data from the data management program are presented. The virtual store enables participants to navigate through the store, choose a product from alternatives, and select or return products. Moreover, consumer-related shopping behaviors (e.g., shopping time, walking speed, and number and type of products examined and bought) can also be collected. The protocol is illustrated with an example of a store layout experiment showing that shelf length and shelf orientation influence shopping- and movement-related behaviors. This demonstrates that the use of a virtual store facilitates the study of consumer responses. The virtual store can be especially helpful when examining factors that are costly or difficult to change in real life (e.g., overall store layout), products that are not presently available in the market, and routinized behaviors in familiar environments.

This paper describes the use of a desktop virtual store to create virtual shopping environments to investigate in-store consumer behavior. A description of the protocol to build and run experiments, example results from an experiment concerning store layout, and important considerations when conducting virtual store experiments are presented.

People&#39;s responses to products and/or choice environments are crucial to understanding in-store consumer behaviors. Currently, there are various ...

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between cell types or even within a given type? What kinds of spatial rules do they follow? The answers to such fundamental questions in vivo have been hampered so far by a lack of tools for high resolution single cell labeling. Here, a detailed protocol to target single cells with a MultiColor FlpOut (MCFO) technique is provided. This method relies on three differently tagged reporters (HA, FLAG and V5) under UAS control that are kept silent by a transcriptional terminator flanked by two FRT sites (FRT-stop-FRT). A heat shock pulse induces the expression of a heat shock-induced Flp recombinase, which randomly removes the FRT-stop-FRT cassettes in individual cells: expression occurs only in cells that also express a GAL4 driver. This leads to an array of differently colored cells of a given cell type that allows the visualization of individual cell morphologies at high resolution. As an example, the MCFO technique can be combined with specific glial GAL4 drivers to visualize the morphologies of the different glial subtypes in the adult Drosophila brain.

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and establish a variety of interactions with their neighbors. This protocol describes how to reveal the morphology of single cells and to investigate cell-cell interaction by using the well-established Gal4/UAS expression system.

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between ...

A Novel Method for Involving Women of Color at High Risk for Preterm Birth in Research Priority Setting

Involvement of patients and the public is now recognized to be essential for the good conduct of research. Patient and public involvement in research priority setting and funding decisions is only beginning to be recognized as important, and methods for doing so are nascent. This protocol describes the Research Prioritization by Affected Communities (RPAC) protocol and findings from its use with women at high socio-demographic risk for preterm birth. The goal was to directly involve these women in identifying and prioritizing their unanswered questions about pregnancy, birth and neonatal care, and treatment so that their views could be included in research priority setting by funders and researchers. The RPAC protocol may be used to meaningfully involve under-represented groups at high-risk for specific health problems, or those who face disproportionate burden of disease, in research strategy and funding priority setting.

This manuscript describes the Research Prioritization by Affected Communities (RPAC) protocol and findings from its use with women at risk for preterm birth. Using the protocol, women identified and prioritized their unanswered questions about pregnancy, birth and neonatal care aimed at influencing research priority setting by funders and researchers.

Involvement of patients and the public is now recognized to be essential for the good conduct of research. Patient and public involvement in research ...

Frame-by-Frame Video Analysis of Idiosyncratic Reach-to-Grasp Movements in Humans

Prehension, the act of reaching to grasp an object, is central to the human experience. We use it to feed ourselves, groom ourselves, and manipulate objects and tools in our environment. Such behaviors are impaired by many sensorimotor disorders, yet our current understanding of their neural control is far from complete. Current technologies for investigating human reach-to-grasp movements often utilize motion tracking systems that can be expensive, require the attachment of markers or sensors to the hands, impede natural movement and sensory feedback, and provide kinematic output that can be difficult to interpret. While generally effective for studying the stereotypical reach-to-grasp movements of healthy sighted adults, many of these technologies face additional limitations when attempting to study the unpredictable and idiosyncratic reach-to-grasp movements of young infants, unsighted adults, and patients with neurological disorders. Thus, we present a novel, inexpensive, and highly reliable yet flexible protocol for quantifying the temporal and kinematic structure of idiosyncratic reach-to-grasp movements in humans. High speed video cameras capture multiple views of the reach-to-grasp movement. Frame-by-frame video analysis is then used to document the timing and magnitude of pre-defined behavioral events such as movement start, collection, maximum height, peak aperture, first contact, and final grasp. The temporal structure of the movement is reconstructed by documenting the relative frame number of each event while the kinematic structure of the hand is quantified using the ruler or measure function in photo editing software to calibrate 2 dimensional linear distances between two body parts or between a body part and the target. Frame-by-frame video analysis can provide a quantitative and comprehensive description of idiosyncratic reach-to-grasp movements and will enable researchers to expand their area of investigation to include a greater range of naturalistic prehensile behaviors, guided by a wider variety of sensory modalities, in both healthy and clinical populations.

This protocol describes how to use frame-by-frame video analysis to quantify idiosyncratic reach-to-grasp movements in humans. A comparative analysis of reaching in sighted versus unsighted healthy adults is used to demonstrate the technique, but the method can also be applied to the study of developmental and clinical populations.

Prehension, the act of reaching to grasp an object, is central to the human experience. We use it to feed ourselves, groom ourselves, and manipulate ...

An Objective and Child-friendly Assessment of Arm Function by Using a 3-D Sensor

Progressive and irreversible muscle atrophy characterizes Spinal Muscular Atrophy (SMA) and other similar muscle disorder diseases. Objective assessment of muscle functions is an essential and important, although challenging, prerequisite for successful clinical trials. Current clinical rating scales restrain the movement abnormalities to certain predefined coarse-grained individual items. The Kinect 3-D sensor has emerged as a low-cost and portable motion sensing technology used to capture and track people&#39;s movement in many medical and research fields. A novel approach using this 3-D sensor was developed and a game-like test was designed to objectively measure the upper limb function of patients with SMA. The prototype test targeted joint movement capability. While sitting in a virtual scene, the patient was instructed to extend, flex, and lift the whole arm in order to reach and place some objects. Both kinematic and spatiotemporal characteristics of upper limb movement were extracted and analyzed, e.g., elbow extension and flexion angles, hand velocity, and acceleration. The first study included a small cohort of 18 ambulant SMA patients and 19 age- and gender-matched healthy controls. A comprehensive analysis of arm movement was achieved; however, no significant difference between the groups were found due to the mismatch of patient&#39;s capability and the test difficulty. Based on this experience, a second version of the test consisting of a modified version of the first game with increased difficulties and a second game targeting muscle endurance were designed and implemented. The new test has not been conducted in any patient groups yet. Our work has demonstrated the potential capability of the 3-D sensor in assessing such muscle function and suggested an objective approach to complement the clinical rating scales.

An objective measure of muscle functions is challenging especially in children. Based on a commercially available digital 3-D sensor, a child-friendly gaming test was developed to assess upper limb function for clinical trials.

Progressive and irreversible muscle atrophy characterizes Spinal Muscular Atrophy (SMA) and other similar muscle disorder diseases. Objective assessment ...

Methods of Pairing and Pair Maintenance of New Zealand White Rabbits (Oryctolagus Cuniculus) Via Behavioral Ethogram, Monitoring, and Interventions

New Zealand White (NZW) laboratory rabbits (Oryctolagus cuniculus), as well as their ancestors the European Rabbit, are a social species that exhibit numerous benefits to being housed accordingly. Although these rabbits are innately gregarious, certain behaviors can still arise when kept in captivity, which if left unchecked, can confound research results or lead to wounding, which in extreme cases can be severe. To prevent these issues, there must be a well-structured plan for the monitoring and maintenance of paired laboratory rabbits. The purpose of this protocol is to present effective procedures for establishing newly paired NZW rabbits as well as methods for successful maintenance. Multiple methods have been tested for the creation of newly paired female rabbits from the vendor, but the most efficacious technique emphasizes capitalizing on the stress bonding from transport, urine marking, pairing in a neutral cage with no forced sharing of resources and a system of monitoring and intervention. To determine the best method of housing paired rabbits in a standard caging environment, data were collected to generate a behavioral ethogram. Behaviors were then quantified as positive, neutral or negative and were tracked across the lifespan of the pair to determine which behaviors indicated pair success or failure. With the newfound knowledge of socially housed laboratory NZW rabbit behavior, enrichment intervention was applied to alleviate aggression and prevent wounding, thus resulting in a higher percentage of successful pairs. Through several years of trialing different pairing methods, the development of the ethogram and the resulting enrichment interventions, understanding of the highly complex social constructs that dominate pair housed rabbit behavior has dramatically increased and allowed for the provision of more species-specific care and increased standards of welfare.

Methods of Pairing and Pair Maintenance of New Zealand White Rabbits Oryctolagus Cuniculus Via Behavioral Ethogram, Monitoring, and Interventions

Though European rabbits are a social species, socially housing them can be challenging. Therefore, there must be a thorough understanding of behaviors and social structures of pair-housed laboratory rabbits. Here we present a protocol to identify pairing methods, species-typical hierarchy establishment behaviors and behaviors that warrant appropriate intervention.

New Zealand White (NZW) laboratory rabbits (Oryctolagus cuniculus), as well as their ancestors the European Rabbit, are a social species that exhibit ...

Using Clicker Training and Social Observation to Teach Rats to Voluntarily Change Cages

Cage cleaning is a routinely performed husbandry procedure and is known to induce stress in laboratory rats. As stress can have a negative impact on well-being and can affect the comparability and reproducibility of research results, the amount of stress experienced by laboratory animals should be minimized and avoided when possible. Further, the direct contact between the rat and animal caretaker during the cage change bears hygiene risks and therefore possibly negatively impacts the well-being of the rats and the quality of the research. 
Our protocol aims to improve the routinely performed cage changing procedure. For this reason, we present a feasible protocol that enables rats to learn via clicker training and observation to voluntarily change to a clean cage. This training helps to reduce stress caused by the physical disturbance and handling associated with the cage changes and concurrently enables a reduction in direct contact between animal and animal caretaker after the training phase is completed.
The implementation of clicker training to rats is fast and easy. Rats are generally interested in the training and efficiently learn the desired behavior, which entails changing cages through a pipe. Even without training, the rats learn to perform the desired behavior by observation, as 80% of the observational learning group successfully changed cages when tested. The training further helps to establish a relationship of trust between trainer and animal. As hygiene and well-being are both very important in animal experiments, this protocol might also help to improve high-quality research.

This protocol introduces a method of cage change for rats via clicker training. Rats learn the desired behavior not only by direct training but also by observational learning. The implementation of this fast and easy protocol might help to improve well-being and hygiene in rodent facilities.

Cage cleaning is a routinely performed husbandry procedure and is known to induce stress in laboratory rats. As stress can have a negative impact on ...

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.

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 ...