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

Summary

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.

Abstract

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's progress was documented. This aids the caretakers so they remain focused on the actions necessary to socialize the animals and limit the animal'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's Culture of Care.

Introduction

The principles of the 3Rs (Replacement, Reduction and Refinement) discussed by Russell and Burch in 1959, form today'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. "Refinement means any decrease in the incidence or severity of inhumane procedures applied to those animals which still have to be used"¹.

A contemporary definition additionally states that refinement is "advancing research into animal welfare by exploiting the latest in vivo technologies and by improving understanding of the impact of welfare on scientific outcomes"². This implies that not only are today'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 time^4,5.

Common to both of these species is that they rank high on the "socio-zoological scale". 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 outcomes^7,8.

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'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 role^13,14. From a research perspective, each individual animal is important to obtain 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 "herd animal" to an "individual". 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 "transition phase" 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 project^16,17. 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's culture of care^21,22,23.

The pigs were housed according to the Institution'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'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 recommendations^25,26, 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.

Protocol

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

1. 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.
2. 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.
3. 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.

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).

1. On the two consecutive days after arrival, introduce the treats.
   1. 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.
2. On day three and successive days introduce familiarizing the pig with "handling by a caretaker". Enter the pen during feeding.
   1. Sit down at arm'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.
   2. 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).
3. 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.
   1. 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.
   2. 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.
4. 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.
5. 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).

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.

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.

Representative Results

Applying the transition phase as described in the protocol'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 research projects within two weeks of arrival and the total number of days was the measure chosen to show the success of the transition.

Based on Checklist 1, the transition phase of 20 pigs has been followed to verify that it is possible to transition pigs from farm pigs to research pigs within two weeks (Figure 4). All pigs completed the list within 12 days, showing that the timeframe set was sufficient.

Checklist 1 was modified based on the first experiences. A new stringent checklist (Checklist 2) was developed. To improve and standardize the transition further, the major revision was increasing the number of daily sessions and limiting the amount of time spent per session. Comparing the two checklists shows that pigs transitioned using Checklist 2 had similar progression during the first days of transition, whereas the later steps were reached quicker. A comparison between the use of Checklist 1 and Checklist 2 can be seen in Figure 5.

After the completion of the transition phase the animals will allow basic outcome measures like taking the temperature and inspection of surgical wounds. When a pig (female) has been transitioned to allow touching of the tail area a urine sample can be collected using an ostomy bag placed around the external genitals but under the anus (Figure 6).

Another example of a data outcome that can be collected is a blood sample. At our facility we often place a venous catheter in an ear vein during surgery. Following recovery, the catheter can be used to administer medicine to reduce the number of intramuscular injections, take multiple blood samples to support the project and evaluate the pig's return to normal physiology (Figure 7).

Moving on from the transition phase the clicker training will build on the transition phase to allow further benefits during handling and to obtain outcome data. Training the pigs to follow a target stick will allow the caretaker to move the pig e.g. onto a scale to monitor bodyweight (Figure 8) or into a transport crate (Figure 9) if the pigs need to be transported to surgery or an imaging facility. For one project, the pigs were trained to stand still while medicine was administered in the eyes (Figure 10).

When starting the clicker training it becomes apparent that each pig is unique and they respond differently to the training. During training, this is acknowledged and training sessions are accommodated accordingly. Some pigs need two or three times as many training sessions to learn the same task, which can be problematic if certain tasks need to be learned at specific time points to support the research project.

Animal data	Checklist 1 (n=20)	Updated checklist (n=20)
Species	Domestic pig (Sus scrofa domesticus)	Domestic pig (Sus scrofa domesticus)
Breed	(Danish Landrace x Yorkshire) x Duroc	(Danish Landrace x Yorkshire) x Duroc
Sex	Female	Female
Weight	35-80 kg (mean 47,5 kg)	40-80 kg (mean 62,5 kg)
Age	10-22 weeks	10-22 weeks

Table 1: Basic characteristics of the pigs used to evaluate the checklists.

Figure 1: The transition phase checklist. As illustrated, several of the activities take place on the same day but at different time intervals. Steps 3-5 takes place during feeding whereas steps 1-2 and 6-7 take place at defined time points during the day. Notice that each session must be performed during the time indicated on the top of the checklist but only takes approximately 2 minutes to carry out per pig. The shaded boxes indicate that the task is not to be performed. The full checklist can be found in supplementary file 1. Please click here to view a larger version of this figure.

Figure 2: Some of the enrichment used at the department Please click here to view a larger version of this figure.

Figure 3: Illustration of the position of the caretaker during steps 2.2 and 2.3 of the protocol. Please click here to view a larger version of this figure.

Figure 4: Timeline of the transition phase using Checklist 1. Please click here to view a larger version of this figure.

Figure 5: Progression of transition phase. A scatterplot showing the progression of the transition phase using Checklist 1 () compared to Checklist 2 (Ο). Each plot corresponds to the observation for one pig. Mean and SD are indicated by the error bars. The y-axis is the number of days. On the x-axis the steps are referred as follows: 1; Apples from the hand. 2; Touch the pig during feeding. 3; Touch the hind and tail during feeding. 4; Touch the hind and tail while giving treats. Please click here to view a larger version of this figure.

Figure 6: Urine sample collection via ostomy bag placed around outer genitals during feeding. The pig remains calm during the procedure and takes no notice of the ostomy bag. Please click here to view a larger version of this figure.

Figure 7: Intravenous dosing during feeding after transition phase. The caretaker is able to sit in close proximity to the animal and focus on infusing the medicine correctly. Please click here to view a larger version of this figure.

Figure 8: Stepping onto scale using target stick. The pig is eager to follow the target stick due to the positive reinforcement training. Please click here to view a larger version of this figure.

Figure 9: Following target stick into transport crate. This procedure is useful if the pig needs to be transported to e.g. an imaging facility. Please click here to view a larger version of this figure.

Figure 10: Pig trained to place its head in a holder and stand still so medicine can be given in the eyes without sedation. After the medicine is given, the pig rests the head on the holder and a visual eye inspection can be performed. Please click here to view a larger version of this figure.

Supplementary File 1. Please click here to download this File. The full Transition Phase Progression Checklist ready-to-print. Note that a change in the arrival date field affects the dates in the sheet.

Discussion

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' commitment to the work increases and that the positive emotions the pigs experience is reflected in their daily behavior.

The arrival of new pigs and the smooth transition to research pig has long been an area of focus but changes have been difficult to introduce. To create an overview of the work and the benefits it was decided to use a checklist to monitor the effectiveness of the prioritized steps. In Checklist 1, the steps were based on actions expected to have most impact on transition. The transition of 20 pigs were scored and evaluated using Checklist 1. Then the list was adjusted to eliminate the encountered problems. These mostly related to misunderstandings and inconsistent reporting. A critical step was the interpretation of each step and which actions the step involved. Hence, the description of each step was elaborated and the checklist was modified to be more intuitive. One step was removed to simplify the distinction between success and failure when touching the pig. To make it clear whether the step was performed or not a checkbox to indicate if the step was performed () was added in addition to a box stating if the pig performed the action (+) or refused to perform the action (÷). To support the modifications additional time was allocated to train the use of Checklist 2. This discussion took place in groups with the aim to discuss each step and agree upon the success criteria. In relation to the practical execution, important issues were raised. For instance, when the pigs are used to the caretakers and treats, they can be very intrusive which can be intimidating and even dangerous to the caretakers. As a last modification, the number of sessions with each pig was increased while the time spent on each session was reduced.

To evaluate Checklist 2, 20 pigs were included. The data for step 4 is missing for three pigs, due to miscommunication between veterinarian and caretaker and underscores the need for communication. The time used daily is estimated to be the same, but the pigs complete the transition phase in fewer days. Having defined certain time slots during the day where the steps needed to be performed demanded a change in some daily routines. In combination with the total time needed to prepare and perform the steps this has been the biggest limitation in the implementation. The checklists contain 7 steps each, but due to the modification of the list the comparison in figure 3 is reduced to four comparable steps. Implementing a thorough structure during the transition phase creates awareness and makes the refinement initiative a priority in a busy research facility.

Implementing a structured checklist has enabled an evaluation of the variation between the individual pigs. It was interesting to notice that for some pigs, the number of days until they understood the treat was often higher compared to completion of the steps related to touching by the caretaker. Another interesting observation was a rather sudden change in the willingness to be touched. One session, the pig would back away instantly, and at the next session only a few hours later the pig would allow for touching for long periods (more than a minute at a time).

To further prepare pigs for research projects, positive reinforcement training is started following the transition phase. A clicker training program is recommended. It allows for several caretakers in shifts to participate in the training of the animals. Using the clicker eases the shift between caretakers because the reward signal remains the same. When the clicker training commences it becomes evident that each pig is unique and responds to the training differently. Within a few sessions, however, all animals are eager to participate in the training sessions and fully enjoy the time spent with the caretaker. Clicker training the pigs enables specific tasks in relation to research projects to be carried out without using force. It also reduces the inter-animal variation seen in some projects e.g. gait analyses. It can also be of great help in the daily chores e.g. when the pig is moved within the research facility or is weighed. A critical step in the clicker training is the level of trust between pig and caretaker when the clicker is introduced. The pig needs to trust the caretaker as it needs to come close but remain focused on the task instead of the caretaker. Another critical step is the choice of treat. The animal must perceive it as a reward. Occasionally we come across a pig that does not find apples tasty and we then swap the apple with e.g. a piece of date or prune. It is important to consider the risk from working with large pigs, especially when they are excited in a training setting. They have a powerful bite, and they can jump up in an attempt to reach treats at unexpected time-points. To counteract these dangers, consider using the target stick to direct the attention away from the trainer, keep the treats clearly out of reach, work in short sessions, and do not run out of treats.

Working with the focused transition and training of pigs has brought with it an increased enthusiasm from the caretaker staff. Education in the field of positive reinforcement training has been a priority and given rise to the fact that caretakers experience a stronger commitment to the research projects. Involvement of the caretakers in the hands-on planning of new projects in dialogue with the researcher and the veterinary staff enables the caretakers to give qualified suggestions on how training could support the research results and the animal welfare. The collection of more data from each pig without causing harm is one of the cornerstones of the 3Rs. Weekly meetings focused on the transition and training have increased the focus on each individual pig and enhanced the department’s Culture of Care. Seeing the pigs when they are about to start a training session is rewarding in itself, and the behavior more than suggests that the pigs enjoy the sessions. When planning the training of pigs for specific projects, bear in mind that the pigs will enter a state of excitement when they get familiar with the training routines. Ideas to counteract this include; If possible, do not train where samples are to be collected. So if you plan to do blood samples in the home pen, try to do the training somewhere else. The pigs will know their daily routine, so plan to take samples at time points where training is not usually performed if possible. When training has to be where the samples are to be collected, let the pig explore the area for a set number of minutes each time, before beginning the training or data collection protocol.

Placing a structure on our transition phase has been an overall positive initiative. A reduction in the stress of the pigs shortly after arrival and a quick transition to having pigs that enjoy the company of the caretakers rather than hurtling about the pens is evident. We have experienced a turnaround in the staff from a point of view where they only saw extra work, to the point where they see the benefits of transitioning the pigs using the checklist. The protocol has an immense application potential and can be applied as an important tool in almost every set up involving research pigs. We now use it to transition both farm pigs and minipigs and aim to implement the clicker training even further in projects related to e.g. cardiovascular science, neuroscience and ophthalmology. The general attitude and Culture of Care in the department has been raised and there is a wish and eagerness to propel these new experiences further to merge research and animal welfare continuously and apply the 3Rs.

Materials

Name	Company	Catalog Number	Comments
Wrap, e-power, blå, 7,5cm	E-vet A/S, Ole Rømers Vej 26 A, DK-6100 Haderslev	203165B	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.
Apples	Varius	Uknown	Used as treats
Klikker (Clicker)	E-vet A/S, Ole Rømers Vej 26 A, DK-6100 Haderslev	TX2289	Clicker used in training