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

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.