The authors thank the animal caretakers for taking care of the rabbits and their compassionate and dedicated work. Thanks to Kai Diederich for proofreading the manuscript.

Positive Reinforcement-Based Training for Routine Rabbit Husbandry Procedures

The authors declare no conflicts of interest related to this publication.

Between 2015 and 2019, more than 49 million animals were used for scientific purposes in the European Union (and Norway); 1,745,037 (3.5 %) of them were rabbits1. Rabbits are mostly used for regulatory research (e.g., quality control, toxicity, and other safety testing, including pharmacology) and routine production of biological substances (e.g., blood-based products)2. Although efforts are made to replace animal experiments with animal-free methods, the use of animals is still necessary for some of these purposes. Whenever an animal experiment cannot be replaced, it is crucial to reduce the number of animals and refine experimental as well as housing and husbandry conditions to minimize suffering. Russel and Burch described this strategy as the 3R principle (replace, reduce, refine) in 19593 and, in 2010, it was implemented into the Directive 2010/63/EU on the protection of laboratory animals used for scientific purposes4. The role of refinement sometimes referred to as &#39;Cinderella&#39; of the 3Rs5, has gained growing importance. While replacement and reduction are long-term strategies towards the ultimate goal of the Directive to fully replace animal experiments, refinement enables the immediate improvement of animal welfare5, which in turn has the potential to improve scientific quality6. Essential parts of refinement are animal handling and training, as pointed out in Annex III of the Directive 2010/63/EU. The Directive stipulates that animal facilities should design habituation and training programs for the laboratory animals adapted to species, procedure, and project4. Appendix A of the ETS No. 123 suggests that the laboratory animal personnel spends time &#34;talking to, handling, training and grooming animals&#34;7.
Some handling procedures cause stress, especially for terrestrial prey animals such as rabbits. Examples are being cornered, restrained (i.e., grabbed by a human, held in the arms of a human, or immobilized by a device), and lifted (i.e., picked up from the floor) for a health inspection or experimental procedures8. A survey focusing on pet rabbits revealed that 57-61% of them struggled when being lifted9,10 and some even showed fear related aggression (i.e., biting)11. This emphasizes the need for gentle handling and training techniques. It has already been demonstrated for other small mammals such as mice that gentle handling techniques decreases anxiety12. Moreover, gentle handling in combination with training reduces stress, anxiety, and depression-like behavior in mice13. In rhesus macaques and chimpanzees, physiological measures related to stress were less affected when they were previously trained on a procedure14,15. It may be assumed that if an animal can choose to participate in the training voluntarily, it can actively engage in and control the situation, which improves the animal&#39;s welfare and may also contribute to more robust and reliable scientific data16.
Many animal training procedures are reliant on operant conditioning principles, particularly the use of positive reinforcement and shaping17,18,19. Operant conditioning requires an animal to elicit behaviors that can then be increased or decreased20. There are four common contingencies that are used to describe operant conditioning: positive and negative reinforcement as well as positive and negative punishment21,22. In rabbits, positive punishment is used when a person approaches (undesired stimulus) an animal to move it away from or towards a particular location23. In the context of handling a rabbit on the examination table, an example for negative reinforcement is the removal of pressure (undesired stimulus) on the rabbit&#39;s body when the rabbit keeps still; an example for negative punishment is withdrawing a food reward (desired stimulus) when the rabbit starts struggling. Neither positive/negative punishment nor negative reinforcement are recommended in animal training. Punishment causes negative emotional affects24, such as distress as shown in dogs25. Instead, positive reinforcement should be the method of choice when training animals25,26. Positive reinforcement training (PRT) means that a desirable stimulus (e.g., a food reward) is provided after the animal showed a desired behavior, which increases the likelihood that the animal will display the desired behavior again in the future27. PRT often involves the application of a conditioned reinforcer. A conditioned reinforcer (secondary reinforcer) usually is a neutral stimulus such as a sound, which the animal learns to associate with an unconditioned reinforcer (primary reinforcer, e.g., food reward)28. After the animal displays the desired behavior, the conditioned reinforcer is immediately presented, followed by the presentation of the food reward26. A well-known example of PRT is clicker training, where a clicker is used for producing a click as a secondary reinforcer26.
The training should follow predictable patterns, which is achieved by designing a training protocol. The training protocol describes how a goal behavior can be reached step by step (i.e., behavioral shaping) while each step is defined by objective criteria and should be easily accomplished26. If the training follows a particular protocol, it is possible that the laboratory animal staff alternates with training the animals. Important to note is that the personnel needs to know the principles of learning and training techniques in order to correctly apply them26. Animal training often incorporates behavioral shaping. Behavioral shaping by hand was deliberately used for the first time by Skinner and colleagues in the early 1940s29. In contrast, in previous work, Skinner rather focused on &#34;making small changes in the physical environment in order to implement a program of successive approximation&#34;29. In an article published in Scientific American, he explained that reinforcing a behavior increases the likelihood that the behavior will be repeated which &#34;makes it possible to shape an animal&#39;s behavior almost as a sculptor shapes a lump of clay&#34;30. Since then, shaping has experienced growing importance for captive animals29,30,31,32. For reproducing and improving shaping protocols, it is crucial to assess the protocols in a systematic and objective manner33,34,35,36.
A recently published systematic review and meta-analysis by Pfaller-Sadovsky et al. found that conditioned reinforcement was most frequently studied in horses and dogs, though cats, cattle, fish, goats, and non-human primates were also investigated37. In laboratory animal science, PRT is widely used for non-human primates38,39,40 and dogs41,42. In addition, pigs43, goats44, and even small mammals, such as mice13 and rats45,46,47, were successfully trained using a clicker in the laboratory setting. To the authors&#39; knowledge, the introduction of PRT for laboratory rabbits has only been rarely reported yet48; existing peer-reviewed literature published several decades ago is outdated and does not meet the criteria of modern ethical research. Although few non-peer-reviewed pieces of information suggest that rabbits can be successfully trained using PRT49, there seems to be a lack of knowledge on suitable training protocols for rabbits that are housed in laboratory animal facilities. The underlying reasons may be varying. For instance, training techniques used may not be described in detail in research articles or data are generated for regulatory research and therefore not being published. Moreover, rabbits may not be the main focus of refinement research since other species such as mice and rats are more frequently used in research. Since rabbits can mask stress and discomfort extremely well, their negative emotional state when being handled may often remain unrecognized. The size of a rabbit allows humans to restrain these animals for procedures while other species such as non-human primates may not be held with the hands for similar interventions without being seriously injured50. However, these facts should not prevent the development or sharing of PRT protocols for laboratory rabbits.
Regarding the potential benefits of PRT, first attempts were made to fill the knowledge gap on suitable training protocols for laboratory rabbits. To facilitate routine procedures such as handling, health inspections, and weighing, training protocols were developed and their feasibility for female New Zealand White rabbits was evaluated. Comparisons were carried out to assess whether the alternative handling technique described in the training protocols was less aversive than the conventional handling technique and whether the training protocols can serve as refinement measures. Figure 1 demonstrates the time schedule of the present study.
Animal maintenance
Thirteen female New Zealand White rabbits were obtained from commercial sources (arrival at animal facility at the age of approximately seven weeks of age). The animals were free of all viral, bacterial, and parasitic pathogens listed in the FELASA recommendations51. Protective clothing and equipment (gloves, masks, hair nets) were worn when conducting the steps of the present protocol. A group of six and a group of seven animals were housed together in a floor pen of 2.8 m &#215; 2.8 m (floor housing), respectively. The floor was covered with fine and flaky wooden bedding material (autoclaved). Moreover, nesting material and paper wool were scattered on top of the bedding. Three plastic houses with two openings each (37 cm &#215; 60 cm; height 30 cm) and a plastic tunnel (length: 58 cm; diameter: 16 cm) served as shelters. Additionally, enrichment items such as wooden gnawing blocks, hay and willow balls, and snack balls filled with food pellets, that fall out when moving the ball, were provided. The rabbits had free access to tap water, pelleted food, and autoclaved hay as well as straw. Further information on food and enrichment items are listed in the Table of Materials. The animal room was cleaned on Thursdays.
The rabbits were maintained under standard conditions (room temperature and relative humidity given as mean &#177; standard deviation: 20.3 &#177; 0.4 &#176;C and 50 &#177; 5 %) on a light:dark cycle of 12:12 h (artificial light with additional daylight incidence). A radio was turned on 30 min before the beginning of the light phase for approximately 8 h to habituate the rabbits to ambient noises.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bed-r&#8217;Nest</td>
			<td>The Andersons, Inc., Maumee, USA</td>
			<td></td>
			<td>The successful implementation of the protocol does not depend on this particular product. You could use comparable products by other companies.&#160;</td>
		</tr>
		<tr>
			<td>Finger Clicker</td>
			<td>ASCO</td>
			<td></td>
			<td>https://ascoshop.de/products/fingerclicker</td>
		</tr>
		<tr>
			<td>G*Power</td>
			<td></td>
			<td>version 3.1.9.7</td>
			<td></td>
		</tr>
		<tr>
			<td>Heu-Proppen</td>
			<td>Ssniff Spezialdi&#228;ten GmbH, Soest, Germany</td>
			<td></td>
			<td>The successful implementation of the protocol does not depend on this particular product. You could use comparable products by other companies.&#160;</td>
		</tr>
		<tr>
			<td>IBM Corp. Released 2020. IBM SPSS Statistics for Windows,&#160; Armonk, NY: IBM Corp</td>
			<td>IBM Corp</td>
			<td>Version 27.0.</td>
			<td>in the manuscript referred to as &#34;software for advanced statistical analysis&#34;</td>
		</tr>
		<tr>
			<td>Kaninchen Haltung + 25 mg/kg Salinomycin-Natrium-Sondermischung</td>
			<td>Ssniff Spezialdi&#228;ten GmbH, Soest, Germany</td>
			<td></td>
			<td>The successful implementation of the protocol does not depend on this particular product. You could use comparable products by other companies.&#160;</td>
		</tr>
		<tr>
			<td>Lignocel block</td>
			<td>Lignocel J. Rettenmaier &#38;S&#246;hne GmbH + Co KG, Rosenberg, Germany</td>
			<td></td>
			<td>The successful implementation of the protocol does not depend on this particular product. You could use comparable products by other companies.&#160;</td>
		</tr>
		<tr>
			<td>Lillebro gesch&#228;lte Sonnenblumenkerne&#8221;, Martina GmbH, M&#252;nchen, Germany) and Rainbow Foraging Bits (Bio-Serv, Flemington, USA)&#160;</td>
			<td>Martina GmbH, M&#252;nchen, Germany</td>
			<td></td>
			<td>The successful implementation of the protocol does not depend on this particular product. You could use comparable products by other companies.&#160;</td>
		</tr>
		<tr>
			<td>Microsoft Excel for Microsoft 365 MSO</td>
			<td>Microsoft Corporation</td>
			<td>Version 2305</td>
			<td>in the manuscript referred to as &#34;spread sheet&#34;</td>
		</tr>
		<tr>
			<td>New Zealand White rabbits&#160;</td>
			<td>Charles River Laboratories France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plattformwaage DE 35K5DL</td>
			<td>KERN &#38; SOHN GmbH, Balingen-Frommern, Germany</td>
			<td></td>
			<td>The successful implementation of the protocol does not depend on this particular product. You could use comparable products by other companies.&#160;</td>
		</tr>
		<tr>
			<td>Premium scientific bedding (fine &#38; flake)</td>
			<td>SAFE, Augy, France</td>
			<td></td>
			<td>The successful implementation of the protocol does not depend on this particular product. You could use comparable products by other companies.&#160;</td>
		</tr>
		<tr>
			<td>Rainbow Foraging Bits</td>
			<td>Bio-Serv, Flemington, USA</td>
			<td></td>
			<td>The successful implementation of the protocol does not depend on this particular product. You could use comparable products by other companies.&#160;</td>
		</tr>
		<tr>
			<td>Snackball f&#252;r Kaninchen &#8211; 7 cm</td>
			<td>TRIXIE Heimtierbedarf GmbH &#38; Co. KG, Tarp, Germany</td>
			<td></td>
			<td>The successful implementation of the protocol does not depend on this particular product. You could use comparable products by other companies.&#160;</td>
		</tr>
		<tr>
			<td>Target stick with integrated clicker</td>
			<td>TRIXIE Heimtierbedarf GmbH &#38; Co. KG, Tarp, Germany</td>
			<td></td>
			<td>The successful implementation of the protocol does not depend on this particular product. You could use comparable products by other companies.&#160;</td>
		</tr>
		<tr>
			<td>Trixi Transportbox Capri Open Top</td>
			<td>TRIXIE Heimtierbedarf GmbH &#38; Co. KG, Tarp, Germany</td>
			<td></td>
			<td>The successful implementation of the protocol does not depend on this particular product. You could use comparable products by other companies.&#160;</td>
		</tr>
		<tr>
			<td>Weiden-Heuball</td>
			<td>JR FARM GmbH, Holzheim-Pessenburgheim, Germany</td>
			<td></td>
			<td>The successful implementation of the protocol does not depend on this particular product. You could use comparable products by other companies.&#160;</td>
		</tr>
	</tbody>
</table>

training laboratory rabbits to refine routine husbandry procedures

Non-aversive handling and training techniques for laboratory animals are required to facilitate experimental and routine husbandry procedures, improving both animal welfare and scientific quality. Clicker training was utilized to develop training protocols for rabbits to refine stressful routine husbandry procedures usually associated with lifting (i.e., being picked up from the floor)/restraining (i.e., being held in the arms of a human) them. Thirteen female New Zealand White rabbits were trained over three weeks. All rabbits learned the predefined goal behaviors: they followed the target stick, jumped onto the weighing scale, entered a transport box, and reared while placing their front paws onto the trainer&#39;s hand. In addition, ten animals jumped from the floor onto the sitting trainer&#39;s lap and allowed the trainer to lift their paws off the surface while sitting on the trainer&#39;s lap. For some individuals, the protocols had to be adapted by additional interim steps. At the end of the training, the rabbits reliably showed the expected goal behaviors, even after short and long training breaks. With few exceptions, a familiar person other than the trainer could elicit the goal behaviors from the rabbits (generalization), though further sessions were required for generalization. In the voluntary approach test, the rabbits preferred interacting with the trainer in the 1st trial but spent as much time with an unfamiliar person as with the trainer in the 2nd trial. The behavioral observations suggested that picking the rabbits up with the transport box, as described in the protocol, instead of restraining them with the scruff of their neck and lifting them on the arm, was less aversive. All in all, the training protocols were feasible and can serve as a refinement strategy in laboratory animal facilities. In the interest of animal welfare, the training protocols should be applied wherever possible.

Watch this Scientific Journal Video about Author Spotlight: Training of Laboratory Animals for Gentle and Stress-Free Handling at JoVE.com

Author Spotlight: Training of Laboratory Animals for Gentle and Stress-Free Handling

Training protocols involving positive reinforcement for routine husbandry procedures in rabbits were developed and proved to be successful, as the rabbits reliably showed the trained behaviors at the weekly general examination. Picking the rabbits up with the transport box was less aversive than the conventional handling technique.

Training Laboratory Rabbits to Refine Routine Husbandry Procedures

Non-aversive handling and training techniques for laboratory animals are required to facilitate experimental and routine husbandry procedures, improving ...

training-laboratory-rabbits-to-refine-routine-husbandry-procedures

Research

JoVE Journal

Behavior

2.2K Views.  German Federal Institute for Risk Assessment. Training protocols involving positive reinforcement for routine husbandry procedures in rabbits were developed and proved to be successful, as the rabbits reliably showed the trained behaviors at the weekly general examination. Picking the rabbits up with the transport box was less aversive than the conventional handling technique. 