Training Laboratory Rabbits to Refine Routine Husbandry Procedures

The training protocols were developed to refine routine procedures such as handling, health inspections, and weighing in laboratory rabbits. Their feasibility was evaluated in this explorative study using thirteen female New Zealand White rabbits. The training protocols could be reliably applied to successfully train the rabbits. Most rabbits were able to learn the goal behaviors defined in the training protocols within less than the 3-week training period, remembered them after 1-week training breaks, and generalized them to another person than the trainer. Few adaptations to the protocol had to be made to meet the need of individual animals. The main finding of the present study was that the refined handling technique for picking up rabbits, as described in the training protocols, was less aversive than the conventional handling technique. Interestingly, the trained rabbits showed their exploratory behavior not only towards the familiar trainer but also an unfamiliar person in a voluntary approach test.

Meaning of the target

The target served as a basis for the present training protocol. When introducing the target, the animals learned that their behavior affected the environment and generated consequences. Introducing the target builds trust between the human and the animal, as demonstrated by rabbit #4, it took 7 training sessions before following the target stick into the training arena but learned the following goal behaviors quickly (i.e., within 1-3 trainings sessions). This rabbit #4 seemed to gained trust in the trainer during the target introduction, which facilitated the training of the other goal behaviors. However, this rabbit still lacked trust in other persons and was not willing to show most of the goal behaviors in the generalization sessions.

It must be noted that the target was not defined as endpoint in some protocol sections. Instead, for example, the animals had to follow the target for a variable number of hops or stay on the scale for a variable number of seconds. Variable rewarding pattern was chosen to increase and maintain the rabbits' attention and motivation in the training.

Signals

After the rabbits had successfully performed step 5.1, in all following training sessions, the trainer placed the target in close distance to the rabbit's nose to signal the rabbit that the training trial started. If the rabbit touched the target with the nose/mouth, willingness to participate in the training was considered and the training began.

The present protocols did not include any further signals, e.g., the scale, the transport box, or the hand were not considered signals. Instead, the target was used to lead the animal into the training arena, onto the scale, into the transport box, into the air (rearing) or onto the trainer's lap. This means that the goal behaviors "weighing", "entering transport box", "rearing", and "jumping on lap" were modifications of "following the target". The reason for this was the requirements for the training protocol: it should be feasible to be carried out by the staff of animal facilities, that may not be experts in animal training, and easily integrated into the daily work routine. Therefore, the protocols must be efficient and achieve the goal behaviors quickly.

However, the hand that is placed in front of the rabbit (step 8.3), the scale or the transport box also have the potential to become both a signal and a target for the paws. Therefore, it may be possible to define further steps that go beyond the present protocols and do not require the use of the target stick. For example, as soon as the rabbit sees the scale, the scale signals the rabbits to jump onto it with all four paws.

Adaptations of the protocols

Although it may be expected that the phenotype of almost genetically identical laboratory animals is very similar, there are behavioral "differences between individuals that are consistent over time and across situations [and] are referred to as personality"⁵⁵, e.g., some animals are rather shy and others bold⁵⁶. It may take shy, less explorative animals longer to learn the defined goal behaviors of the protocol (e.g., rabbit #4). Even if standard training protocols may be suitable for the majority, training has to consider the needs of an individual animal and adapt to it, when necessary²⁶. Thus, additional smaller interim steps should be defined for those rabbits that fail to learn a training step of the protocol. The additional interim training steps should help them to successfully proceed towards the following step. Especially because training plans are developed in theory and then tested in practice, it may become apparent that additional training steps are required to train a particular goal behavior.

With respect to the present training protocols, some interim training steps had to be added for the goal behaviors "following the target", "entering transport box", "rearing", and "jumping on lap", as described in detail in the section on the representative results since individual animals failed to continue with the following training step. Interestingly, the interim steps 8.2+ and 9.3+ were required by most rabbits, emphasizing their importance. Therefore, these steps were added as a note to the training protocols.

Alternatively, to the defined step 8.2+, the trainer could place a hand on the floor with the palm facing upwards and let the rabbit step on the hand with one and eventually both front paws. Moreover, it is conceivable that a rabbit requires additional interim steps before step 9.3+, e. g., first the rabbit places one hind paw and after that both hind paws onto the trainer's lap. If a rabbit does not accept to be touched in steps 9.5-9.9, the duration of touching the different body parts could be gradually increased. Moreover, initially only the index and middle fingers could be used to stroke the animals with feather-light pressure. Then the number of fingers used for touching and the pressure may be increased in several steps.

Refinement of handling techniques

It can be assumed that handling is highly aversive, especially in rabbits with a shy personality trait. Rabbits are terrestrial prey animals and being lifted (i.e., picked up from the floor) or restrained (i.e., held in the arms of a human) for a health inspection and experimental procedure is likely to cause anxiety and distress. One option to refine handling methods was introduced in the training protocols: instead of restraining the rabbit by the scruff of the neck and lifting the rabbit onto the other arm, the rabbit can be led with the target stick into the box and carried in the box to the examination table, as previously suggested^50,57, where the rabbit is gently transferred from the box to the table. Alternatively, the top of the box can be removed, or the rabbit can be guided with the target stick out of the box.

The behavioral observations performed after the rabbits were picked up using either the conventional handling technique or the transport box revealed that the conventional handling technique was associated with more stress- and anxiety-related behavior than the alternative technique. This conclusion was drawn from the high number of animals refusing the food reward on the examination table and the increased time duration spent hiding in the shelters when the conventional handling technique was applied. Interestingly, the handling technique did not affect the time of interaction with the trainer, indicating that the handling process may have not impaired the human-animal-relationship. However, besides the trainer, the transport box also served as "handling device" (i.e., the handling device of the conventional technique was the trainer, the handling devices of the alternative technique were both the trainer and the transport box). Therefore, the time of interaction with the transport box was also analyzed. Although the rabbits had just been picked up with the transport box, they spent more time interacting with the box or in the box than when being picked up by the conventional technique. This suggested that the transport box could be associated with a positive affect. When comparing the duration spent interacting with both "handling devices" (i.e., sum of time duration spent interacting with the box and the trainer), the box handling technique increased the time duration interacting with the "handling devices", i.e., this technique had a clear positive effect on the behavioral response of the rabbits towards the "handling devices" in anticipation of being handled. Similar findings were made in laboratory mice, demonstrating that gentle handling techniques and training reduced stress and anxiety: animals that were picked up using a tunnel or the cupped hands spent more time voluntarily interacting with the "handling device" and showed less anxiety-related behavior than tail-handled mice¹². Training appeared to strengthen this effect. Leidinger et al. demonstrated that gentle-handled (tunnel/cup) mice showed less urination, defecation, and vocalization when being restrained by the scruff of their neck and less floating behavior in the Morris Water Maze Test when they were trained using PRT in comparison to untrained mice¹³.

Feasibility of the protocols for daily work – training duration, retention, and generalization

The number of training sessions required to learn a goal behavior depended on the individual animal and the goal behaviors, which comprised a different number of steps ranging from section 3 to section 9 of the protocol presented above.

According to the number of animals that succeeded in learning a goal behavior and the number of adaptations that had to be made to the protocols for the individual animals, the training steps had different difficulty levels. The behavior "following the target" was the first training experience of the rabbits and, therefore, it took longer for some animals to train on this behavior than the other goal behavior, as discussed above. While all animals could be trained on "weighing" according to the present protocol, additional interim steps were required for "entering transport box" by two animals and for "rearing" as well as "jumping on lap" by almost all rabbits. This may be explained by the degree of physical contact between animal and trainer which was necessary for performing the different goal behaviors. "Weighing" and "entering transport box" did not involve any physical contact between animal and human. The final step of "rearing" (8.3) and the steps 9.2-9.4 of "jumping on lap" required the rabbits to make physical contact with the trainer. Additionally, the trainer touched the rabbit's shoulder, back, rump, ears, or paws in steps 9.5-9.9 of "jumping on lap", increasing the degree of physical contact between animal and trainer. Accepting being touched was more challenging for some rabbits than for others. As discussed above, it was beneficial to adapt the training protocols of these goal behaviors and add interim steps.

However, three rabbits required additional training sessions to complete the goal behavior "jumping on lap" (#4, #7, #10). Physical impairments that prevented these animals from jumping onto the trainer's lap could be excluded. Moreover, the rabbits were able to jump onto their houses, the heights of these were higher than the trainer's lap.

It was noticeable that two of these three rabbits (#4 and #10) needed longer to get used to the trainer as well as the food reward in the habituation sessions and partially needed more training sessions to reach the final step of other goal behaviors. This was also true for rabbit #7, which had an injury at the tail due to fights within the group since the fourth habituation session. Therefore, rabbit #7 was picked up, transferred to the examination table, treated, and inspected daily for 15 days, which may have resulted in increased caution towards humans. On the one hand, it is crucial to note that the training performance can be affected by, for instance, an impaired health status, increased stress levels, or a disturbed human-animal relationship due to other interventions. On the other hand, these observations demonstrated the importance of habituation to the trainer and the food reward. Some animals may require longer to get familiar with the presence of the trainer and accept the food reward from the trainer. If additional habituation sessions had been conducted, rabbit #4 and #10 may have performed better in the training sessions. The relevance of habituating rabbits to humans has already been reported in the literature. The commercial breeder, from which the rabbits used in this study were purchased, recently introduced regular petting sessions, which reduced the stress-related behavior and eliminated aggressions⁵⁸. In an earlier study, petting and handling of young rabbits were demonstrated to decrease anxiety-related behavior, increase their weight gain, and reduce the mortality rate⁵⁹.

Once the rabbits had learned the different goal behaviors, they were still able to display them after two short (one week) and a long (approximately 7.5-9.5 weeks) training breaks, except from individual exceptions. These observations may indicate that it is sufficient to ask the rabbits to perform the trained goal behaviors once a week when the general examination is performed, and the room/pen is cleaned.

For the feasibility of the protocol for daily work life, it is also crucial that the rabbits generalize the trained goal behaviors to other persons than the trainer. In case of vacation or illness, another person has to continue the training and care for the animals. The majority of rabbits (twelve out of thirteen) generalized the goal behaviors "weighing", "entering transport box", and "rearing" to the animal caretaker, although, in some cases, it took the rabbits more than one training sessions to show the desired behavior. "Weighing" seemed to generalize best; followed by "entering transport box" and "rearing". However, "jumping on lap" appeared to be more difficult to generalize to another person. It took the animals longer to demonstrate the desired behavior (9.9.) and two rabbits, that previously learned the final step 9.9, failed to show this goal behavior, possibly due to the higher physical contact needed (see above). One rabbit (#4) did not show any steps of the goal behaviors "weighing", "entering transport box", and "rearing" and was not interested in interacting with the animal caretaker. Rabbit #4 was one of the animals that needed longer to get used to the trainer and the food reward in the habituation sessions. This may indicate that, for some individuals, additional sessions in which the animal caretakers spend time with the rabbits, habituate them to accept the food reward from them and build up a positive bond are beneficial²³. Rabbits may be able to distinguish between different persons, as demonstrated for rats⁶⁰. Further factors, that have the potential to affect the rabbits' performance in the sessions with the trainer and the animal caretaker may be unconscious signals the trainer gave the animals during the training sessions, e.g., the position of the target stick or reward bowl, the body position or movement. If the trainer is not aware of these signals, they will not be documented and the other persons will not mimic these signals.

Furthermore, the training skills of the persons working with the animals, including the routine of handling the target stick and reward presentation, can influence the behavior of the animals. Besides knowledge of learning principles and animal communication, practical experiences are needed to successfully train an animal⁶¹ and retrieve a goal behavior. Since generalization sessions involving a skilled and an unskilled person were not compared, the relevance of the factor "training skills" cannot be determined. However, to the authors' knowledge, a large proportion of the laboratory animal staff is not trained in animal training and thus the conditions under which the generalization sessions were performed (i.e., involving an animal caretaker with only little previous experiences in clicker training) represented a realistic setting in laboratory animal facilities.

The representative data of the protocols provide animal facilities an idea of the workload associated with training the different goal behaviors. The data may help animal facilities to create their own work schedules when implementing (parts of) the protocol. Depending on the individual animal and goal behaviors, some additional time should be reserved for the generalization process. However, the time duration may vary when working with male rabbits or another rabbit breed. In the animal facilities, discussion should be held on whether appropriate time exists and what needs to be changed to allow time for animal training.

Besides the time required for training the animals, it must be considered that training needs skilled trainers and teaching the staff how to train animals can also be time-consuming. If training was a mandatory part of the education of the laboratory animal personnel, i.e., animal caretakers, veterinarians, and researchers, the implementation of animal training in facilities would be facilitated and would take less time. According to Annex III of the Directive 2010/63/EU, "establishments shall set up habituation and training programs suitable for the animals, the procedures and length of the project", i.e., animal training is required by law. In Germany, such programs must be submitted when applying for an animal testing license. This requires that persons planning animal experiments and working with laboratory animals are skilled in animal training. However, at least in Germany, animal training is not part of the training schedule of animal caretakers in the field of research and clinic^62,63,64. Moreover, it is not explicitly mentioned in the EU recommendations for education and training framework⁶⁵. Therefore, as long as training has not been implemented in these regulations, continued professional development focusing on positive reinforcement training needs more attention. The laboratory animal personnel need to learn the principles of learning and training techniques, the needed criteria, the transfer of procedure between trainers, record keeping, and how to respond to animals that are not participating in a desired manner to ensure consistency and predictability.

Human-animal-interaction

The intensive human-animal-interaction-time during the training sessions provides an optimal setting for strengthening the relationship between the individual rabbits and the trainer^26,66. A positive human-animal-relationship is beneficial for the animals' well-being since it can reduce their stress levels in husbandry and experimental settings⁶⁷. In the present training protocols, the trainer considers the agency of the rabbits and they can decide to participate (or not participate) in the training on a voluntary basis. Providing the animals with agency is beneficial for their well-being ¹⁶ and should therefore be pursued in experiments as well as housing and husbandry. There are different behavioral agency levels⁶⁸; participating in PRT may be attributed to the level of action driven agency, i.e., "actively behaving to achieve current outcomes [e.g., procure food]"⁶⁸.

When analyzing the voluntary approach of the rabbits towards the trainer and an unfamiliar person, an intensive human-animal-interaction could be observed. The results revealed that all rabbits, except from one animal in the 1^st trial (#12), interacted with both the trainer and the unfamiliar person. They appeared to recognize the trainer since they showed a clear preference for interacting with the trainer in the 1^st trial and some of them jumped with all four paws onto the trainer's lap; a behavior that was not displayed towards the unfamiliar person. Although the rabbits spent more time interacting with the trainer than the unfamiliar person in the 1^st trial, the interaction time did not differ anymore in the 2^nd trial. A reason for this observation may be that the rabbits expected to receive a food reward from the trainer in the 1^st trial and, due to lack of food rewards in the voluntary approach test, they did not spend more time with the trainer in the 2^nd trial anymore. These data nevertheless emphasized that the trained rabbits quickly habituated to the presence of an unfamiliar person and none of them was too shy to interact. In contrast, they were very explorative and interested in the unfamiliar person. However, since an untrained control group was not investigated, the effect of training on the voluntary approach behavior of the rabbits is unclear.

A hypothesis explaining the behavior of rabbit #12 in the 1^st trial is that this individual still had to habituate to the presence of the unfamiliar person in the 1^st trial and was more interested in interacting with both the trainer (70 seconds) and the unfamiliar person (9 seconds) in the 2^nd trial. Rabbits have different personality traits which may affect the level of comfort around unfamiliar persons and in turn the latency to approach or the interaction time duration with a person^69,70.

Limitations

We are aware that all rabbits in the pen/room, not only the one participating in the training can hear the click (i.e., the conditioned reinforcer) during a training session and its reinforcing effect may be weakened since the food reward (i.e., natural/unconditioned reinforcer) is not presented. If a rabbit outside the training arena shows a trained behavior and hears the click, but no food is presented (and this scenario occurs several times), the trained behavior may be extinguished. This phenomenon is referred to as extinction^64,71. However, since all goal behaviors of the present protocols depend on the target presented by the trainer, extinction is unlikely to occur in our training setting.

Since the walls of the training arena were transparent and the training arena was located in the pen, the rabbits could see, hear, and smell each other during the training, which is a clear welfare-related advantage. Moreover, the rabbits may have observed their group members during the training sessions and learned from each other. In rats, it was previously shown, that individuals learned to perform a goal behavior by observing their cage mate being trained by PRT⁴⁵. This fact may be considered as both an advantage and disadvantage. The learning progress of the animals may be faster when they can observe each other. However, the data of the individual animals may be influenced by the performance of the group members and may not be attributed solely to the training of the individual animal. Thus, training animals without any contact with their group members or single-housed rabbits could take longer.

Behavioral observations of the individuals (in the voluntary approach test and after applying the different handling techniques) were carried out in their familiar social environment and group to avoid stress and distraction caused by a novel environment and separation from their group, which in turn would have affected the behavioral observations. The disadvantage is, however, that the rabbits may have influenced each other's behavior during the observation sessions.

The effects of the handling techniques were only investigated in seven animals involving the trainer as person handling the animals. The authors are planning a follow-up study including an a priori sample size calculation to reproduce the findings with other handlers and to further examine the differences between the handling techniques.

All humans involved in training, handling, and the voluntary approach test were female. It was not investigated whether the training performance of the rabbits or their behavior would vary if male persons were involved.

The present training protocols should give the reader an idea of what can be trained in rabbits but do not comprise all potential goal behaviors that would be beneficial to train in a laboratory rabbit. For instance, marking a rabbit as suggested in the protocol section on general requirements would also be worth training using PRT.

As pointed out further above, the choice of the right reward is not trivial. Due to hygiene restrictions, the use of fresh herbs as food rewards is not allowed in some laboratory animal facilities and, therefore, less appropriate food rewards such as sunflower seeds or grain-based foraging treats must be used. With respect to the decision on the food reward, some rules and regulations of current laboratory practices are detrimental to animal well-being and effective training and, therefore, may need to be reconsidered on a case-by-case basis. It may also be possible to use alternatives to food rewards, e.g., gentle touches or interactions with the trainer or enrichment items. However, the trainer must make sure for each individual animal that the alternative is to reward the animal because a reward is only a reward if the rabbit really favors it. Especially touches may not be received as rewarding by all rabbits.

Conclusions

In summary, the present study was a first step for filling the knowledge gap on suitable training protocols for laboratory rabbits. Female New Zealand White rabbits could be trained for routine husbandry procedures using PRT and generalized the goal behaviors learned to other persons, though additional time should be reserved for the generalization process. Goal behaviors without physical contact seemed to be easier to learn than behaviors that required physical contact to humans. After the animals successfully completed the training of a goal behavior, it was sufficient to retrieve them once a week in the context of the general examination and pen cleaning. Training protocols considered to be generally applied to all rabbits are convenient, but the trainer must be aware that, in some cases, it must be adapted to the needs of the animal. The results on handling techniques suggested that picking the rabbits up with the transport box instead of restraining them by the scruff of their neck and lifting them on the arm was less aversive. All in all, the present protocols provide feasible instructions for the habituation and training process of rabbits and can serve as refinement in laboratory animal facilities.

Animals must not suffer due to saving labor, time or money (at least according to the German Animal Welfare Act⁷²). It is important to care for and handle all animals in the gentlest way possible. Since untrained animals suffer to a higher degree from anxiety and distress caused by experimental and routine husbandry procedures than their trained counterparts¹³, training is a simple refinement measure to effectively prevent them from experiencing negative affective states.