Name: methods of pairing and pair maintenance of new zealand white rabbits (oryctolagus cuniculus) via behavioral ethogram, monitoring, and interventions
Uploaded: 2018-03-16T14:30:00
Description: Watch this Scientific Journal Video about Methods of Pairing and Pair Maintenance of New Zealand White Rabbits Oryctolagus Cuniculus Via Behavioral Ethogram, Monitoring, and Interventions at JoVE.com

The authors would like to thank the multiple laboratories at the U of M that utilize rabbit models for the generous use of their colonies, in particular, the laboratory of Dr. Eugene Chen and the Center for Advanced Models for Translational Sciences and Therapeutics (CAMTraST) team. Thanks also to the U of M ULAM husbandry team, Jenny Jones for enrichment coordination and Katie Wearsch for assistance with pairing.

This protocol followed the standards of care developed and utilized by the Unit for Laboratory Animal Medicine (ULAM) of the University of Michigan (U of M), which receives approval and oversight of all animal care procedures and methods by the IACUC. The IACUC is in full compliance with the eighth edition of the Guide. All rabbits were housed in cages that were a minimum of .46 m2 per cage which exceeds Guide minimum requirements.
1. Methods for the Creation of Female Pairs ...

     

<ol>
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One of the most difficult challenges facing a rabbit social housing program is the differentiation between species typical aggressive or communication behaviors and problematic fighting behaviors. An in-depth and extensive knowledge of normal rabbit social behavior is required of all husbandry and veterinary staff monitoring the pairing process since many behaviors that appear to be a fight behavior are actually necessary hierarchy establishment behaviors that typically do not result in injury. For example, chasing and f...

The regulations governing laboratory animal care provide well-defined recommendations regarding rabbit social housing. Two of the three primary standards used by AAALAC International to evaluate animal care and use programs describe guidelines for species-appropriate husbandry and housing. The first of these guidance resources, the 8th edition of the Guide for the Care and Use of Laboratory Animals1 (Guide) states that social species such as rabbits should only be singly housed as an exception and single housing should not be the housing standard. The second, the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes2, states that rabbits are an inherently social species that should be housed in accordant social situations with the exception of qualifying veterinary or research related reasons. The Office of Laboratory Animal Welfare (OLAW) also mandates that single housing is only appropriate when there is scientific justification that has been evaluated and approved of by the Institutional Animal Care and Use Committee (IACUC), the animals are incompatible or the veterinary staff has a health or welfare concern3.
In addition to meeting regulatory compliance, peer-reviewed literature provides many examples of the physical and psychological benefits of pair housing NZW rabbits. In order to provide the highest possible standards of animal welfare, species-appropriate housing must be provided while meeting the specific goals of the researcher. Socially housed NZW rabbits engage in more active behaviors4, display amplified fitness and show diminished levels of gastrointestinal stasis due to increased locomotor behaviors5. Physical activity is particularly important for laboratory rabbits as they are prone to osteoporosis4 when housed in a cage environment. In addition to the physical benefits of social housing, there is a myriad of psychological benefits. Pair-housed NZW rabbits display increased species-specific activities6, receive enrichment benefits from social stimulation4, show increased rates of self-soothing activities7 as well as affiliative behaviors such as allogrooming8 and exhibit an increased ability to manage anxiety from novel stimuli, i.e. stress buffering5. Additionally, in comparison to socially isolated NZW rabbits, socially housed NZW rabbits did not differ in immune function9 or antibody production10, however, singly housed rabbits did have higher heart rates11 and increased white blood cell counts10 when compared to socially housed rabbits.
Just as the benefits of social housing are well documented in the literature, so are the detriments of social isolation. Single housing of a social species can lead to physiological indicators of stress12. Singly housed NZW rabbits spend increased amounts of time being inactive and performing abnormal behaviors4 and stereotypic behaviors indicative of frustration13,14,15. Common stereotypes include: chewing on metal such as cage bars or feeder/waterer, excessive auto-grooming, licking of the cage and self-inflicted negative behavior16. If they must be housed singly, additional attention should be paid to counter-balance the stress from social isolation. The Guide recommends providing additional housing space, only housing singly for the minimum amount of time possible, allowing for occasional time in a larger housing situation such as a floor pen and providing additional and varied enrichment opportunities including interaction with other rabbits when possible1. AAALAC International additionally recommends providing protected access such that animals can have visual, auditory and olfactory contact with other animals17.
Given the choice, rabbits prefer to be with other rabbits. Preference testing demonstrated that female NZW rabbits worked almost as hard for limited social contact as they did for food18. Multiple other studies have shown that paired rabbits, when given the choice, were observed in physical contact with each other 4,8,19,20. These studies highlight the significant value rabbits place on socialization with conspecifics. Interestingly, these results do not apply to only female rabbits. When provided with a modified cage divider which was half opaque, half clear with small holes drilled into the clear portion to allow for enhanced but protected social access, male NZW rabbits spent more time in the quarter of the cage that provided the most access to a neighboring male than males in cages without the modified dividers. The male NZW rabbits with protected social access were significantly more active, spending more time in activity and rest behaviors and less on maintenance behaviors such as grooming which is associated with boredom and social deprivation. Males with a modified divider were also less fearful of unfamiliar staff as demonstrated by a reduced latency to approach an unfamiliar hand than males with a standard divider; possibly due to the social buffering effects of interaction with a partner21. Additionally, the circadian patterns of activity and rest of the male rabbits with modified dividers became synchronized in a crepuscular pattern, similar to wild rabbits, whereas socially isolated rabbits varied significantly from one another in their patterns of activity and rest21. Literature suggests that male rabbits are not ideal candidates for social housing due to aggression post sexual maturity14,22,23,24. In wild populations, females are more gregarious than males6 and multiple females often share a living space while males tend to maintain a larger distance between other males which can lead to increased aggression when placing males in the limited space of a laboratory cage15,25. Some studies have attempted to socially house males in a larger pen setting with mixed results14,22,26,27. One study attempted pairing males in a cage setting which resulted in all three pairs being separated for aggression14. The following protocol is the first to the authors&#39; knowledge that allows for housing males with sibling males successfully in a cage setting into adulthood. There is no pairing of unrelated males or adult males due to the possibility of aggression and no male/female pairings due to the research uses of the colony.
The first step towards rabbit social housing is the establishment of new pairs. This article describes the methods that allowed for the greatest success within this institution of establishing new female pairs and the ethogram used for structured monitoring. The protocol utilizes methods such as urine marking6,28, stress bonding, providing neutral territory and assuring no forced resource sharing. In wild rabbit colonies, the dominant buck urinates on the rabbits of his breeding group (including kits, juveniles and does) thereby scent marking the rabbits that belong in his social group29. This idea has been translated to the laboratory setting by applying freshly collected urine from a male rabbit to the forehead of newly paired females. This imbues the females with a shared scent to indicate to them that they are within the same breeding group and they do not need to compete aggressively. An investigation of using urine marking in creating new female rabbit pairs found that unmarked pairs were more aggressive and exhibited less affiliative behaviors than pairs marked with buck urine6. Stress bonding has also been shown to be an effective method of pairing in the companion rabbit community by creating a mildly stressful situation that both rabbits are mutually exposed to in order to encourage bonding30. This is typically achieved by a car ride or a walk in a carrier. A theory is that the rabbits will carry over the feelings of security that they received from having a partner during this stressful time into their future bonding experiences and they, therefore, will be more likely to form a successful pair31. Pairing new arrival rabbits that have gone through the stress of transport from the vendor creates a natural stress bonding experience that can be capitalized on to assist with the pairing. Once pairs are established, an effective monitoring program must be implemented to track pair behavior over time in order to intervene when necessary. This article highlights the behavioral ethogram that was developed to enable a complete understanding of rabbit social behavior and methods for pair maintenance. Pair maintenance is often a point of difficulty in facilities due to a lack of comprehensive literature describing standard rabbit social interactions and what they may indicate. Rabbit pairs are often separated for interactions that appear to be fighting behavior but are actually normal social interactions that can be monitored and assuaged with proper intervention. This article looks to alleviate this by providing a broad list of behaviors to track and what they may be indicative of. Additionally, environmental enrichment intervention is utilized to aid in the prevention of pair failure. Adequate environmental enrichment may decrease fearful behaviors and allow for better coping with stressors1, reduce abnormal behaviors24,32, provide opportunities for essential physiological requirements33 and overall increase welfare34. The results of previous investigation at this institution showed that increasing environmental enrichment facilitates greater success in pair housing by decreasing the aggressive behaviors that arise around the age of sexual maturity (12-17 weeks)35. This method has a two-fold benefit; the enrichment creates a distraction to prevent fighting, as well as, allowing for positive interactions through species-typical behaviors.

<table>
	<tbody>
		<tr>
			<td>Euro Rabbit Housing</td>
			<td>Allentown, Inc.</td>
			<td>RBEURO-06</td>
			<td>Middle divider removed 
			http://www.allentowninc.com/large-animal-housing/rabbit-ferret/</td>
		</tr>
		<tr>
			<td>R-Suite Enriched Rabbit Housing</td>
			<td>Techniplast</td>
			<td></td>
			<td>2 attached single cages with middle divider removed 
			http://www.tecniplast.it/en/product/r-suite-rabbit-housing-rack.html</td>
		</tr>
		<tr>
			<td>Lab Animal Marker - Standard Tip</td>
			<td>Ketchum Manufacturing</td>
			<td></td>
			<td>Blue 
			http://www.ketchum.ca/products/lab-animal/animal-markers/1313/animal-markers-standard-tip</td>
		</tr>
		<tr>
			<td>Western Timothy Hay&#160;</td>
			<td>Oxbow Animal Health&#160;</td>
			<td></td>
			<td>http://www.oxbowanimalhealth.com/products/type/detail?object=1530</td>
		</tr>
		<tr>
			<td>Spray Bottles</td>
			<td>Uline</td>
			<td>S-18404BLU</td>
			<td>https://www.uline.com/Product/Detail/S-18404BLU/Spray-Bottles/24-oz-Blue-Spray-Bottles</td>
		</tr>
		<tr>
			<td>Rabbit liners</td>
			<td>Blue Ridge Tissue</td>
			<td></td>
			<td>Custom Versa-Pak T.1503 30&#39;&#39; X 30&#39;&#39; 
			http://www.blueridgetissue.com/products/#versa_pak</td>
		</tr>
		<tr>
			<td>Premium Full Leather Glove</td>
			<td>Wells Lamont</td>
			<td>0-53300-07006-8</td>
			<td>https://www.wellslamont.com/catalog/product/premium-full-leather-glove/</td>
		</tr>
		<tr>
			<td>Various Enrichment Toys</td>
			<td>Bio-Serv</td>
			<td>Multiple</td>
			<td>https://www.bio-serv.com/category/Rabbit_Enrichment_Devices.html</td>
		</tr>
		<tr>
			<td>Fruit and Veggie Medley Dried Treats</td>
			<td>Bio-Serv</td>
			<td>F7227</td>
			<td>https://www.bio-serv.com/Rabbit_Enrichment_Treats/FVM.html</td>
		</tr>
		<tr>
			<td>White Noise Machine</td>
			<td>HoMedics</td>
			<td></td>
			<td>https://www.amazon.com/HoMedics-SS-2000G-Relaxation-Machine-Nature/dp/B00A2JBMRE/ref=redir_mobile_desktop/134-0048439-0235364?_encoding=UTF8&#38;keywords=white%20noise%20machine&#38;qid=1503569413&#38;ref_=mp_s_a_1_6_a_it&#38;sr=8-6</td>
		</tr>
		<tr>
			<td>Safari Soft Bristle Cat Brush&#160;</td>
			<td>Safari&#160;</td>
			<td></td>
			<td>https://www.amazon.com/Safari-Soft-Bristle-Brush-Cats/dp/B0002RJMB4</td>
		</tr>
		<tr>
			<td>Crink-l&#39;Nest</td>
			<td>The Andersons Lab Bedding</td>
			<td>CNK</td>
			<td>http://www.andersonslabbedding.com/irradiated/crink-lnest/</td>
		</tr>
		<tr>
			<td>Manzanita Wood Gnawing Sticks</td>
			<td>Bio-Serv</td>
			<td>W0016</td>
			<td>http://www.bio-serv.com/Rabbit_Enrichment_Devices/W0016.html</td>
		</tr>
		<tr>
			<td>Wood Gnawing Blocks Certified</td>
			<td>Bio-Serv</td>
			<td>K3511</td>
			<td>Small 
			http://www.bio-serv.com/Rabbit_Enrichment_Devices/Wood_Blocks.html</td>
		</tr>
		<tr>
			<td>Paper Bags</td>
			<td>Duro</td>
			<td></td>
			<td>Sack Lunch Bags 
			https://www.amazon.com/Duro-Paper-Lunch-Brown-Count/dp/B01N0AS7QO/ref=sr_1_9?ie=UTF8&#38;qid=1511194982&#38;sr=8-9&#38;keywords=paper+bags</td>
		</tr>
		<tr>
			<td>Various Sources of Cardboard</td>
			<td>Recycled supply boxes from the vivarium&#160;</td>
			<td></td>
			<td>All cardboard is autoclaved prior to use</td>
		</tr>
	</tbody>
</table>

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.

Over a 12-month period, 172 NZW laboratory rabbit pairs (62% female, 38% male) were maintained following the above protocol. Of these pairs, only 20% of male pairs and 1% of female pairs had to be separated for fighting as shown in Figure 5. The pairs that were separated for fighting behaviors were separated at an average of 18.5 weeks old. The majority of total pairs of both sexes (63%) were separated for research purposes that were not related to pairing, w...

Watch this Scientific Journal Video about Methods of Pairing and Pair Maintenance of New Zealand White Rabbits Oryctolagus Cuniculus Via Behavioral Ethogram, Monitoring, and Interventions at JoVE.com

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.

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 ...

The overall goal of this process is to present effective methods for establishing newly paired New Zealand white rabbits, as well as a behavioral ethogram and strategies for successful paired maintenance. This method can help answer key questions in the rabbit social housing field by clearly defining behaviors characteristic to paired laboratory rabbits and defining what those behaviors indicate. The main advantages of this technique include methods for successfully pairing female laboratory animal rabbits in a cage setting and also strategies for successful maintenance of both male and female rabbit pairs.Generally, individuals new to this method will struggle, because we are instinctively inclined to immediately separate any animals engaging in aggressive behavior. However, some agnositic displays are necessary for rabbits to successfully establish their dominance hierarchy. If we intervene too quickly, we disrupt this process, which can delay or prevent the creation of a successful pair.Visual demonstration of this method is critical, as behaviors noted during pairing and pair maintenance can be difficult to learn without direct observation and can appear as fight behaviors when they're actually normal means of social communication. When female NZW rabbits arrive from vendors unpaired, use the following protocol to pair them. First, collect fresh urine from a male rabbit.Overturn the liner underneath its cage so that the plastic side is facing up and the urine pools on the top of the liner. Then, syringe off the urine and store in sterile conical tube. Next, prepare a clean, neutral cage without a center divider.Include at least two low value enrichment items, such as toys or chew sticks, and include two high value destructible enrichment items. Include at least two hideouts, such as a perch, hut, or box, and importantly, include two separate access points for food and water and two separate piles of loose hay. Now, unpack the first female from the shipping container, weigh her, and trim her nails.Then, if needed, mark the rabbit on the tips of the ears with blue or purple nontoxic animal safe ink for individual identification. Next, using a gauze pad or cotton ball, apply approximately one milliliter of the buck urine to the rabbit's forehead and place her into the neutral cage. Then, prepare another female in the same manner, but without the earmark and place her in the same cage.Now, monitor the pair continuously for at least an hour while filling out the rabbit social introduction log. Record each behavior observed at each time point. When biting or fighting behaviors occur, intervene with a squirt of water from the spray bottle.Also use the spray bottle if both rabbits are chasing each other for over 15 seconds, but not if one of the rabbits is fleeing, which is a normal behavior. If intervention is needed, then temporarily separate the rabbits and check each for wounds. If there is any wounding near the eyes, genitals, or any actively bleeding wounds, then do not reintroduce the pair and promptly notify veterinary personnel.If the pair is stable after one hour of observation, then make two more 10 minute observations later in the day. If the pair is still behaving aggressively, continue monitoring them for another hour and reevaluate. If the pair requires multiple interventions and are continuously engaged in aggressive behaviors, increase monitoring and intervention and watch closely for any wounding.If the pair is stable during the two 10 minute checks, leave them paired overnight, and on the following morning, give each a rabbit a physical exam to check for wounds, especially around the genitals. If no wounding is observed, perform daily 10 minute checks for the remainder of the week. If the pair is maintained for a week, then consider the pair stable.Pairs are considered a failure if they demonstrate specific types of wounding, as outlined in the text, or if they are unable to stay together overnight without continuous new minor lesions occurring within one to two weeks. Other pairs can be created using the same method, including female sibling pairs at weaning, female non-sibling pairs within a week after weaning, female weanlings with their mother, male siblings at weaning, or adults that were purchased pre-paired from the vendor. For these types of pairings, a quick five minute observation is all that is needed to ensure a compatible pair.When filling out the social housed interaction and enrichment log, there are a wide variety of behaviors to be familiar with. Several of these affiliative behaviors, and others, are positive indicators of pairing. The establishment of a clear dominant and submissive animal is affiliative.These behaviors include one rabbit chasing, mounting, chin marking, or thumping, while the other accepts the behavior submissively. Sharing resources, such as food, water, and hay, is a positive indicator of pair stability. Another positive behavior is allogrooming, in which one rabbit grooms the other.If the submissive grooms the dominant, this is not a sign of pair breakdown. Observations of self-grooming are also positive. This indicates that the rabbit feels secure in the shared environment.If the two are sharing space or choosing to be in the same location, this is also positive. Another positive behavior is when a rabbit is engaged alone with an enrichment item, or when the pair is interacting with environmental enrichment items together in a non-aggressive, non-competitive manner. When making an observation, note that if no interactions are seen, this is considered a neutral outcome.This does not indicate incompatibility per se, but if the rabbits consistently ignore one another, it could suggest a dominance hierarchy has not yet been established and a high potential for negative behavior. Negative behaviors include fighting or an inability to establish a dominance hierarchy. Fight behavior is defined by both animals performing negative behaviors at the same time, such as the two rabbits circling or chasing each other.Rabbits were examined by veterinary personnel after these altercations and no wounding was observed. Watch closely for biting, wounding, circling, resource guarding, or lunging. When any of these behaviors are observed, intervene with a squirt of water from the spray bottle to distract the rabbits from the behavior.Typically, the rabbits will separate to opposite sides of the cage to groom the water off of their fur. If the water does not cause them to disengage in negative behaviors, put on a pair of heavy gloves and manually separate the pair. There are a variety of natural ways that rabbits use to communicate their dominance hierarchy with each other.When observing these behaviors, place the pair on an increased monitoring status, and for intervention, provide high value enrichment items. Chasing and fleeing are very common in pairs establishing dominance and this behavior does not warrant intervention. Urine spraying is most frequently seen in paired adult males, but can be observed in paired females.Urine may be sprayed on the cage or on the submissive rabbit. Both rabbits have observable urine staining on their fur, as well as on the cage. This pair consists of two adult males, 1.6 years old, who have a stable and well-established dominance hierarchy.The pair is often observed demonstrating mounting, urine staining, grooming, chase flee behaviors, allogrooming, as well as laying together. The urine staining is under close and continuous monitoring and neither male has demonstrated skin irritation or lesions as a result. Mounting is a very typical dominance display, with the dominant mounting and the submissive accepting.Barbering, hair loss as a result of overgrooming by a cagemate is usually first observed on the nose, in between the ears, or on the back of the neck. Monitor this to ensure it does not progress to lesions. This rabbit has observable facial barbering and is under continued monitoring, but has developed no lesions as a result of the barbering.Thumping is done for multiple social communication reasons, one of which is to display dominance. But it might also be sign of submission when accompanied by fleeing. Over a 12 month period, 344 NZW rabbits, 62%of which were female, were paired as described.Of these pairs, only 8%were separated for aggression. The separated pairs were 18 1/2 weeks old on average and only two rabbits had wounding that impacted their utility for further investigations. At the time of separation, for research purposes, the oldest male pair was 75 weeks old, and the oldest female pair was 92 weeks old.Neither pair were wounded and both performed affiliative behaviors. After watching this video, you should have a good understanding of how to create new rabbit pairs, what behaviors to monitor to maintain successful pairs, and how to properly intervene to help a pair sustain a healthy dominance hierarchy. Rabbits can significantly benefit from social housing.As with all socially housed animals, wounding is possible, so all pairing activities should be closely observed by trained personnel with close veterinary oversight. Any lesions should be promptly reported to veterinary personnel.

Research

JoVE Journal

Behavior

Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction

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Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST)

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station INBEST

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Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases

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Moderate Prenatal Alcohol Exposure and Quantification of Social Behavior in Adult Rats

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Infant Auditory Processing and Event-related Brain Oscillations

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The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

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The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

Basic Methods for the Study of Reproductive Ecology of Fish in Aquaria

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A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

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Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities

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Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

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