The research presented here was supported by Charles River and Tufts Cummings School of Veterinary Medicine. The authors are most grateful to Megan Rock and Miranda Gallo for their work with TINT testing.

Both of the following protocols are most accurate when animals are housed on solid bottom caging with bedding material.
1. Nest Scoring6
<ol>
	<li>Weigh out 8-10 g of nesting material.
	<ol>
		<li>Crinkled paper nesting material (such as Enviro-Dri) is recommended as the substrate for nest scoring since most mice build better nests with this substrate6 but other materials can be used9.</li>
	</ol>
	</li>
	<li>Identify the cage(s) to be scored.
	<ol>
		<li>Animals should be housed in solid-bottomed cages for optimal performance on this test.</li>
	</ol>
	</li>
	<li>Place paper nesting material into the cage.
	<ol>
		<li>Nesting material can be provided at any time convenient to the researcher or animal care staff</li>
	</ol>
	</li>
	<li>Return the next day at 7-9 hr&#160;after lights on for scoring. 
	Note: This is the best time to score nests since mice should be inactive10, nests score highest11, and animal rooms are quiet. If animals are active they may trample the nest, altering the score. Variation in nest building will occur depending on the amount of time the mice are given to build, making it important to score the same day and time after new material is provided.</li>
	<li>Use the nest scoring protocol by Hess et al.6&#160;to score each nest (Figure 1).
	<ol>
		<li>First determine whether the material has been manipulated by the mice. This includes being processed or moved around the cage.
		<ol>
			<li>If the material is untouched the whole nest receives a score of 0;</li>
		</ol>
		</li>
		<li>Next determine if the majority of the nesting material has been gathered to a central nest site.
		<ol>
			<li>No nest site: If it is not clear where the nest site is located the cage receives a score of 1 (i.e. the material is spread throughout the cage);</li>
			<li>Nest site: If a centralized nest site is present, consider the nest as if it were a square. Each of the 4 sides of the square can be given a separate score from 2-5. The 4 scores are then averaged for the cage nest score.
			<ol>
				<li>2 = the nest is flat with no shallow walls;</li>
				<li>3 = a slightly cupped shape where the wall of the nest is less than &#189; the height of a dome that would cover a mouse;</li>
				<li>4 = a wall that is &#189; the height of a dome;</li>
				<li>5 = walls that are taller than &#189; the height of a dome, which may or may not fully enclose the nest.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
Note: Alternatively, scoring can occur 3-4 days after the provision of new material, when peak scores are often reached (unpublished data).
2. Time to Integrate into Nest Test12
Note: This test will be most accurate if the animal has a long fiber paper nesting material and has built a nest with a score of at least 2 (see above).
<ol start="6">
	<li>Take a 2 in&#160;square of cotton nesting material (such as a Nestlet) and cut it into quarters, resulting in 4-1 in testing squares.</li>
	<li>Test animals within the first 3 hr&#160;of vivarium lights turning on.
	<ol>
		<li>This window of testing takes advantage of increased nest building behaviors prior to daytime inactivity11.</li>
	</ol>
	</li>
	<li>Open the cage lid and place 1 testing square on the opposite side of the cage from the main nest site.
	<ol>
		<li>Place the test square where it can be seen from outside the cage.</li>
		<li>The test material should be placed in the same location each time the test is conducted.</li>
	</ol>
	</li>
	<li>Return to the cage after 10 min.</li>
</ol>
Note: If the test square is missing from its original location, the cage is TINT positive. If the test square remains in the original location, the cage is TINT negative and additional observations may be needed to determine if the mice require veterinary attention.

<ol>
	<li>Baumans, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Science-based+assessment+of+animal+welfare:+laboratory+animals.">Science-based assessment of animal welfare: laboratory animals.</a> Revue Scientifique Et Technique-Office International Des Epizooties. 24, 503-513 (2005).</li><li>Langford, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coding+of+facial+expressions+of+pain+in+the+laboratory+mouse.">Coding of facial expressions of pain in the laboratory mouse.</a> Nat. Methods. 7, 447-452 (2010).</li><li>Aubert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sickness+and+behaviour+in+animals:+a+motivational+perspective.">Sickness and behaviour in animals: a motivational perspective.</a> Neurosci. Biobehav. Rev. 23, 1029-1036 (1999).</li><li>Latham, N., Mason, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+house+mouse+to+mouse+house:+The+behavioural+biology+of+free-living+Mus+musculus+and+its+implications+in+the+laboratory.">From house mouse to mouse house: The behavioural biology of free-living Mus musculus and its implications in the laboratory.</a> Appl. Anim. Behav. Sci. 86, 261-289 (2004).</li><li>Brown, R. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+behavior,+reproduction,+and+population+changes+in+the+house+mouse+(Mus+musculus+L).">Social behavior, reproduction, and population changes in the house mouse (Mus musculus L).</a> Ecol. Monogr. 23, 217-240 (1953).</li><li>Hess, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Home+improvement:+C57BL/6J+mice+given+more+naturalistic+nesting+materials+make+better+nests.">Home improvement: C57BL/6J mice given more naturalistic nesting materials make better nests.</a> J. Am. Assoc. Lab. Anim. Sci. 47, 25-31 (2008).</li><li>Gaskill, B. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+or+insulation:+Behavioral+titration+of+mouse+preference+for+warmth+or+access+to+a+nest.">Heat or insulation: Behavioral titration of mouse preference for warmth or access to a nest.</a> PLoS ONE. 7, (2012).</li><li>Roper, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nesting+material+as+a+reinforcer+for+female+mice.">Nesting material as a reinforcer for female mice.</a> Anim. Behav. 21, 733-740 (1973).</li><li>Gaskill, B. N., Garner, J. P., Pritchett-Corning, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+reallocation+to+breeding+performance+through+improved+behavioral+thermoregulation.">Energy reallocation to breeding performance through improved behavioral thermoregulation.</a> J. Am. Assoc. Lab. Anim. Sci. 50, 771 (2011).</li><li>Gaskill, B. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+nesting+material+on+mouse+body+temperature+and+physiology.">Impact of nesting material on mouse body temperature and physiology.</a> Physiol. Behav. 110, 87-95 (2013).</li><li>Jirkof, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+postsurgical+distress+and+pain+in+laboratory+mice+by+nest+complexity+scoring.">Assessment of postsurgical distress and pain in laboratory mice by nest complexity scoring.</a> Lab Anim. , (2013).</li><li>Rock, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-to-Integrate+to+Nest+Test+as+an+indicator+of+wellbeing+in+laboratory+mice.">Time-to-Integrate to Nest Test as an indicator of wellbeing in laboratory mice.</a> J. Am. Assoc. Lab. Anim. Sci. 53, 1-5 (2014).</li><li>Gaskill, B. N., Lucas, J. R., Pajor, E. A., Garner, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Working+with+what+you've+got:+Changes+in+thermal+preference+and+behavior+in+mice+with+or+without+nesting+material.">Working with what you've got: Changes in thermal preference and behavior in mice with or without nesting material.</a> J. Therm. Biol. 36, 1193-1199 (2011).</li><li>Aubert, A., Goodall, G., Dantzer, R., Gheusi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+lipopolysaccharide+on+pup+retrieving+and+nest+building+in+lactating+mice.">Differential effects of lipopolysaccharide on pup retrieving and nest building in lactating mice.</a> Brain Behav. Immun. 11, 107-118 (1997).</li><li>Arras, M., Rettich, A., Cinelli, P., Kasermann, H. P., Burki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+post-laparotomy+pain+in+laboratory+mice+by+telemetric+recording+of+heart+rate+and+heart+rate+variability.">Assessment of post-laparotomy pain in laboratory mice by telemetric recording of heart rate and heart rate variability.</a> BMC Vet. Res. 3, (2007).</li><li>Leach, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+effects+of+ovariohysterectomy+and+oral+administration+of+meloxicam+in+laboratory+housed+rabbits.">Behavioural effects of ovariohysterectomy and oral administration of meloxicam in laboratory housed rabbits.</a> Res. Vet. Sci. 87, 336-347 (2009).</li><li>Matsumiya, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+mouse+grimace+scale+to+reevaluate+the+efficacy+of+postoperative+analgesics+in+laboratory+mice.">Using the mouse grimace scale to reevaluate the efficacy of postoperative analgesics in laboratory mice.</a> J. Am. Assoc. Lab. Anim. Sci. 51, 42-49 (2012).</li><li>Bult, A., Lynch, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nesting+and+fitness:+Lifetime+reproductive+success+in+house+mice+bidirectionally+selected+for+thermoregulatory+nest-building+behavior.">Nesting and fitness: Lifetime reproductive success in house mice bidirectionally selected for thermoregulatory nest-building behavior.</a> Behav. Genet. 27, 231-240 (1997).</li><li>Berry, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+natural+history+of+the+house+mouse.">The natural history of the house mouse.</a> Field Stud. 3, 219-262 (1970).</li><li>Gaskill, B. N., Winnicker, C., Garner, J. P., Pritchett-Corning, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+naked+truth:+breeding+performance+in+outbred+and+inbred+strains+of+nude+mice+with+and+without+nesting+material.">The naked truth: breeding performance in outbred and inbred strains of nude mice with and without nesting material.</a> Appl. Anim. Behav. Sci. 143, 110-116 (2013).</li><li>Lee, C. T., Wong, P. T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+effect+and+strain+differences+in+the+nest-building+behavior+of+inbred+mice.">Temperature effect and strain differences in the nest-building behavior of inbred mice.</a> Psychon. Sci. 20, 9 (1970).</li><li>Lee, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+nest-building+behavior+in+inbred+mice.">Development of nest-building behavior in inbred mice.</a> J. Gen. Psychol. 87, 13 (1972).</li><li>Van Oortmerssen, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+significance+genetics+and+evolutionary+origin+of+variability+in+beahvior+within+and+between+inbred+strains+of+mice+Mus+musculus+a+behavior+genetic+study.">Biological significance genetics and evolutionary origin of variability in beahvior within and between inbred strains of mice Mus musculus a behavior genetic study.</a> Behaviour. 38, 1-92 (1971).</li><li>Schneider, J. E., Lynch, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+a+common+physiological+mechanism+underlying+progesterone-induced+and+maternal+nesting+in+mice,+Mus+musculus.">Investigation of a common physiological mechanism underlying progesterone-induced and maternal nesting in mice, Mus musculus.</a> J. Comp. Psychol. 98, 165-176 (1984).</li><li>Lisk, R. D., Pretlow, R. A., Friedman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+stimulation+necessary+for+elicitation+of+maternal+nest-building+in+mouse+(Mus+musculus).">Hormonal stimulation necessary for elicitation of maternal nest-building in mouse (Mus musculus).</a> Anim. Behav. 17, 730-737 (1969).</li></ol>

Brianna N. Gaskill and Kathleen R. Pritchett-Corning are employees of Charles River Laboratories who breeds the mouse strain used in this article.&#160;

Based on these results, nest building is altered depending on the degree of thermal stress, the amount of aggression present within the cage, when animals experience a general malaise, and gathering behavior is not seen in postsurgical and potentially painful mice. Since any and all of these factors will reduce a mouse&#39;s welfare, we believe that nest building can be utilized to identify cages or animals where interventions need to be made to improve their overall well-being. We believe that nest scoring may be a method best utilized for scientific purposes because the scale is more refined and can detect smaller differences than the binary TINT. However, we believe that the TINT is a more practical tool that is best applied during daily husbandry or postsurgical identification of painful animals.
Unlike social predatory species, where displays of illness or injury may be beneficial, displaying signals of weakness in prey species may be the difference between survival or death. Rabbits, for example, have been observed to respond to pain or distress by remaining motionless, especially in the presence of a human, making behavioral indicators of distress seemingly useless16. Ongoing pain behaviors in mice are also difficult to identify and use to quantify pain17. This is especially difficult in nocturnal laboratory species where, at a glance, distressed and inactive mice can easily be confused with sleeping ones.
Resources, such as food or materials which allow for nest building, are strongly tied to the survival and fitness of wild mice5,18,19. In studies, nesting material is a resource laboratory mice are strongly motivated to obtain7,8. Enviro-Dri has been shown to be an ethologically relevant enrichment for mice which allows them to alleviate thermal stress by building more insulating nests7,10,13. The long fiber length, which requires little to no processing, allows even poor nest builders to build better nests6,7. This material is also suitable for nude mice20, which are often susceptible to eye lesions. While Enviro-Dri is superior for nest building, other material (i.e. Nestlets) functions better for the TINT. The presence or absence of the test square from the designated area is less subjective than attempting to determine if a portion of Enviro-Dri strips was removed.
Both nest scoring and TINT can easily be utilized in singly housed animals however issues may arise if 1 animal out of the group may be experiencing pain or distress. The data presented in this manuscript illustrate that thermal stress and cage aggression can be identified in group housed mice. Further work looking into social cohesion and interactions between healthy and unhealthy animals is currently being investigated. Therefore we caution utilizing these methods in group housed animals to detect illness or pain in a subset of the group.
Variation in building and gathering material between types of laboratory mice is well documented7,21-23 and should be considered before using this technique for all types of mice. Specifically, C3H mice do not gather or build as complex nests as other inbred strains21,22. Therefore, normal nest conformation and TINT outcomes in unmanipulated mice should be observed prior to utilizing this technique in any strain or stock. It is possible that different cage types may influence building outcome. For instance, increased convective heat loss experienced in individually ventilated cages may increase overall nest scores due to additional thermal stress.
The utility of the examination of nesting behavior has only begun to be explored. The literature indicates that nests change due to the hormonal status of the dam24,25 and therefore could also be a tool for identifying periparturient females without disturbing them. Although it has not yet been directly tested, researchers studying models of human disease might also use evaluation of nesting behavior as a humane end point or to identify disease onset, ultimately alleviating overall suffering. Alteration in this essential and complex behavioral sequence might also be a more valid, mouse-oriented, task for models of mental health disorders or even drug testing.

Measurement of animal welfare is a difficult and often subjective assessment. When animals are used for scientific purposes, there is an obligation to provide the best care possible and minimize discomfort1. Species-specific objective methods are needed to measure overall well-being for scientific and husbandry purposes. Current methods of welfare assessment in laboratory mice are either time consuming, require expensive equipment, or must be analyzed later to determine if an alteration has occurred2-3. These techniques are useful in a scientific setting, but difficult to incorporate in day to day husbandry. A simple, mouse-oriented method that can quickly and easily identify mice in pain or distress would be a step toward the immediate identification and alleviation of those stressors.
Nests are used by wild mice to shelter from predators, retreat from harsh environmental conditions, and protect young4. Behaviors associated with nest building are therefore highly linked to the survival of wild mice5. Laboratory mice, even though removed from wild conditions for many generations, are highly motivated to build a nest when given proper materials6,7. Any changes to these highly motivated behaviors, or the nest itself, should indicate a substantial alteration in the environment or the animals themselves.
The goal of this method is to exploit highly motivated nesting behaviors in the laboratory mouse to quickly identify pain, illness, or other stressors that may affect or reduce the animal&#39;s overall welfare. This binary cage-side assessment technique is advantageous over other existing methods because little training is needed, testing materials are found in the average mouse colony, and the test can be completed with little to no manipulation of the animals. Additionally, the provision of fresh and or different nesting material on a frequent basis may provide a positive interaction between mice and caretakers since nesting material has been found to be rewarding to mice8.

<table><tbody><tr><td>Enviro-Dri</td><td>Shepherd Specialty Papers</td><td></td><td>Crinkled paper nesting material </td></tr><tr><td>Nestlet</td><td>Ancare</td><td></td><td>Compressed cotton nesting material</td></tr></tbody></table>

nest building as an indicator of health and welfare in laboratory mice

The minimization and alleviation of suffering has moral and scientific implications. In order to mitigate this negative experience one must be able to identify when an animal is actually in distress. Pain, illness, or distress cannot be managed if unrecognized. Evaluation of pain or illness typically involves the measurement of physiologic and behavioral indicators which are either invasive or not suitable for large scale assessment. The observation of nesting behavior shows promise as the basis of a species appropriate cage-side assessment tool for recognizing distress in mice. Here we demonstrate the utility of nest building behavior in laboratory mice as an ethologically relevant indicator of welfare. The methods presented can be successfully used to identify thermal stressors, aggressive cages, sickness, and pain. Observation of nest building behavior in mouse colonies provides a refinement to health and well-being assessment on a day to day basis.

Temperature:
Nests built by groups of 3 C57BL/6 mice were scored in cages held at three different ambient temperatures (20, 25, or 30 &#176;C; Figure 213) after 24 hr. As the temperature increases, nest score decreases. The nest walls lower and nests become more open, allowing for more heat loss.
Aggression:
Groups of male mice with a high level of aggressive interactions which result in wounding can be identified by nest shape. Over a 5 week experiment, nest score was found to negatively correlate with the average number of wounded C57BL/6 male mice in a cage (F1,26 = 13.5; P=0.0011; Figure 3). The specific substrain of C57BL/6 mice did not significantly explain wounding data (F2,26 = 2.69; P = 0.086). Cages with higher scoring nests had fewer mice wounded than those in lower scoring nests regardless of substrain.
Sickness:
Mice experiencing a general malaise have been found to engage in little to no nest building behavior when forced to build a nest14.
Pain:
Nest conformation has been observed to be altered after a painful surgical procedure11,15. The number of animals with a negative TINT increase by approximately 60% after a painful surgery12 but mice returned to positive baseline TINT levels 3 days postoperatively (Figure 4).
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/51012/51012fig1highres.jpg" src="/files/ftp_upload/51012/51012fig1.jpg" width="500px" /> 
Figure 1. Nest scoring diagram. A nest, with internal cavity, that depicts different scoring wall heights6. The figure is reproduced with permission from the Journal of the American Association of Laboratory Animal Science. <a href="https://www.jove.com/files/ftp_upload/51012/51012fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/51012/51012fig2highres.jpg" src="/files/ftp_upload/51012/51012fig2.jpg" width="500px" /> 
Figure 2. Affect of temperature on nest score. Average nest score of mice confined to an ambient temperature of 20, 25, or 30 &#176;C13. The diagonal line indicates a significant linear contrast. The figure is reproduced with permission from Elsevier. <a href="https://www.jove.com/files/ftp_upload/51012/51012fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/51012/51012fig3highres.jpg" src="/files/ftp_upload/51012/51012fig3.jpg" width="500px" /> 
Figure 3. The effect of aggression on nest scores. This graph depicts the correlation between the average nest score of a cage with 5 male mice and the number of those males that were wounded. This was assessed in 3 different substrains of C57BL6 mice. <a href="https://www.jove.com/files/ftp_upload/51012/51012fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="6in" fo:src="/files/ftp_upload/51012/51012fig4highres.jpg" src="/files/ftp_upload/51012/51012fig4.jpg" width="500px" /> 
Figure 4. The effect of pain on TINT. The LSM and SE of a negative TINT are depicted before and after two surgeries12. The figure is reproduced with permission from the Journal of the American Association of Laboratory Animal Science. <a href="https://www.jove.com/files/ftp_upload/51012/51012fig4highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about Nest Building as an Indicator of Health and Welfare in Laboratory Mice at JoVE.com

Nest Building as an Indicator of Health and Welfare in Laboratory Mice

We demonstrate the utility of nest building behavior in laboratory mice as an indicator of welfare. Nest scoring is a sensitive technique that is altered by temperature, illness, and aggression. The time to integrate into nest test (TINT) is a simple cage-side assessment that can detect postoperative pain.

The minimization and alleviation of  suffering has moral and scientific implications. In order to mitigate this  negative experience one must be able to ...

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26.5K Views.  Charles River. We demonstrate the utility of nest building behavior in laboratory mice as an indicator of welfare. Nest scoring is a sensitive technique that is altered by temperature, illness, and aggression. The time to integrate into nest test (TINT) is a simple cage-side assessment that can detect postoperative pain.

Video: Nest Building as an Indicator of Health and Welfare in Laboratory Mice

A Protocol for Housing Mice in an Enriched Environment

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent studies demonstrate that environmental enrichment decreases adiposity, increases energy expenditure, resists diet induced obesity, and causes cancer remission and inhibition in mice. EE typically consists of larger living space, a variety of &#8216;toys&#8217; to interact with, running wheels, and can include a number of other novel environmental changes. All of this fosters a more complex social engagement, cognitive and physical stimulations. Importantly, the toy location and type of toy is changed regularly, which encourages the mice to adapt to a frequently changing and novel environment. Many variables can be manipulated in EE to promote health effects in mice. Thus these approaches are difficult to control and must be properly managed to successfully replicate the associated phenotypes. Therefore, the goal of this video is to demonstrate how EE is properly set up and maintained to assure a complex, challenging, and controlled environment so that other researchers can easily reproduce the protective effects of EE against obesity and cancer.

Environmental enrichment for mice requires a complex and challenging setup, as well as comprehensive husbandry and handling techniques to assure robust metabolic and anti-cancer effects in the mice. This protocol provides detailed procedures to reproduce the above mentioned effects in mice.

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent ...

Assessing Burrowing, Nest Construction, and Hoarding in Mice

Deterioration in the ability to perform "Activities of daily living" (ADL) is an early sign of Alzheimer's disease (AD). Preclinical behavioural screening of possible treatments for AD currently largely focuses on cognitive testing, which frequently demands expensive equipment and lots of experimenter time. However, human episodic memory (the most severely affected aspect of memory in AD) is different to rodent memory, which seems to be largely non-episodic. Therefore the present ways of screening for new AD treatments for AD in rodents are intrinsically unlikely to succeed. A new approach to preclinical screening would be to characterise the ADL of mice. Fortuitously, several such assays have recently been developed at Oxford, and here the three most sensitive and well-characterised are presented.
Burrowing was first developed in Oxford13. It evolved from a need to develop a mouse hoarding paradigm. Most published rodent hoarding paradigms required a distant food source to be linked to the home cage by a connecting passage. This would involve modifying the home cage as well as making a mouse-proof connecting passage and food source. So it was considered whether it would be possible to put the food source inside the cage. It was found that if a container was placed on the floor it was emptied by the next morning., The food pellets were, however, simply deposited in a heap at the container entrance, rather than placed in a discrete place away from the container, as might be expected if the mice were truly hoarding them. Close inspection showed that the mice were performing digging ("burrowing") movements, not carrying the pellets in their mouths to a selected place as they would if truly hoarding them.6



Food pellets are not an essential substrate for burrowing; mice will empty tubes filled with sand, gravel, even soiled bedding from their own cage. Moreover, they will empty a full tube even if an empty one is placed next to it8.



Several nesting protocols exist in the literature. The present Oxford one simplifies the procedure and has a well-defined scoring system for nest quality5. 



A hoarding paradigm was later developed in which the mice, rather than hoarding back to the real home cage, were adapted to living in the "home base" of a hoarding apparatus. This home base was connected to a tube made of wire mesh, the distal end of which contained the food source. This arrangement proved to yield good hoarding behaviour, as long as the mice were adapted to living in the "home base" during the day and only allowed to enter the hoarding tube at night.

Burrowing, nesting, and hoarding are species-typical activities that mice readily perform in the laboratory. This article describes how they can be easily and cheaply assessed. These protocols are extremely sensitive to mouse strain, brain lesions and diseases. Moreover they constitute &#x201C;environmental enrichment&#x201D; for the mice, and embody the &#x201C;Refinement&#x201D; aspect of the &#x201C;3 Rs&#x201D;18.

Deterioration in the ability to perform "Activities of daily living" (ADL) is an early sign of Alzheimer's disease (AD). Preclinical behavioural screening ...

Neuroscience

Olfactory Behavioral Testing in the Adult Mouse

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible circuitry. In addition, this area's unique ability to generate new neurons throughout an organism's lifetime makes it an attractive system for developmental and regenerative biologists alike. Such interest necessitates a means for a quick, yet reliable assessment of olfactory function. Many tests of olfactory ability are complex, variable or not specifically designed for mice. Also, some tests are sensitive to memory deficits as well as defects in olfactory abilities, confounding obtained results.
Here, we describe a simple battery of tests designed to identify defects in olfactory sensitivity and preference. First, an initial general health assessment allows for the identification of animals suitable for further testing. Second, mice are exposed to various dilutions of scents to ascertain whether there is a threshold difference. Third, mice are presented with various scents, both attractive and aversive, that allow for the assessment of olfactory preference. These simple studies should make the initial characterization of olfactory behavior accessible for labs of varied resources and expertise.

Fundamental, yet unique properties of the rodent olfactory system have led to its increasing study among biologists. A relatively simple assessment of its function is then also needed. Here we describe sensitive tests for the characterization of mouse olfactory sensitivity and preference.

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible ...

Biology

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal experimentation whenever possible. When there is no alternative to animal experimentation, the total number of laboratory animals used should be the minimum needed to obtain valuable data. Moreover, appropriate refinement measures should be applied to minimize pain, suffering, and distress accompanying the experimental procedure. The categories used to classify the degree of pain, suffering, and distress are non-recovery, mild, moderate, or severe (EU Directive 2010/63). To determine which categories apply in individual cases, it is crucial to use scientifically sound tools.
The well-being-assessment protocol presented here is designed for procedures during which general anesthesia is used. The protocol focuses on home cage activity, the Mouse Grimace Scale, and luxury behaviors such as burrowing and nest building behavior as indicators of well-being. It also uses the free exploratory paradigm for trait anxiety-related behavior. Fecal corticosterone metabolites as indicators of acute stress are measured over the 24-h post-anesthetic period.
The protocol provides scientifically solid information on the well-being of mice following general anesthesia. Due to its simplicity, the protocol can easily be adapted and integrated in a planned study. Although it does not provide a scale to classify distress in categories according to the EU Directive 2010/63, it can help researchers estimate the degree of severity of a procedure using scientifically sound data. It provides a way to improve the assessment of well-being in a scientific, animal-centered manner.

We developed a protocol to assess well-being in mice during procedures using general anesthesia. A series of behavioral parameters indicating levels of well-being as well as glucocorticoid metabolites were analyzed. The protocol can serve as a general aid to estimate the degree of severity in a scientific, animal-centered manner.

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal ...

Novel Object Recognition and Object Location Behavioral Testing in Mice on a Budget

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various physiological or pathological changes. The object location task (OLT) and the novel object recognition task (NORT) are two effective behavioral tasks commonly used to reveal the function and relative health of specific brain regions involved in memory. While both of these tests exploit the inherent preference of mice for the novelty to reveal memory for previously encountered objects, the OLT primarily evaluates spatial learning, which relies heavily on hippocampal activity. The NORT, in contrast, evaluates non-spatial learning of object identity, which relies on multiple brain regions. Both tasks require an open-field-testing arena, objects with equivalent intrinsic value to mice, appropriate environmental cues, and video recording equipment and the software. Commercially available systems, while convenient, can be costly. This manuscript details a simple, cost-effective method for building the arenas and setting up the equipment necessary to perform the OLT and NORT. Furthermore, the manuscript describes an efficient testing protocol that incorporates both OLT and NORT and provides typical methods for data acquisition and analysis, as well as representative results. Successful completion of these tests can provide valuable insight into the memory function of various mouse model systems and appraise the underlying neural regions that support these functions.

Here we provide a protocol which includes comprehensive instructions for the economical establishment of murine object location and novel object recognition behavioral testing, including the design, cost, and construction of required equipment as well as execution of behavioral testing, data collection, and analysis.

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various ...

Nest Building Behavior as an Early Indicator of Behavioral Deficits in Mice

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents synthetic and/or natural materials (such as twine, tissue, cotton, paper, and hay) as a gauge of their overall well-being and as an ancillary assessment to predict the possible decline in cognition. Typically, changes in nesting behaviors, such as failure to create a nest, indicate a change in health or welfare. In addition, nesting behavior is sensitive to many environmental and physiological challenges, as well as many genetic mutations underlying pathological disease states. The following protocol describes a nesting behavior paradigm that explores the usage of four types of nesting material. In addition, the protocol utilizes intraclass correlations to demonstrate that inter-rater reliability is higher when nests are constructed out of shredded paper compared to other common nesting materials such as cotton squares, paper twists, and soft cob bedding. The chosen methodology and statistical considerations (i.e., intraclass correlation) for this assay may be of interest for those conducting experiments assessing the quality of living of mice.&#160;

Here, we present a protocol to quantify nest building behavior in mice, which is known to be impaired in several neurological disorders and diseases. This protocol examines the utility of four materials and offers the opportunity to quantify the rater agreement in scoring, improving the validity and reliability of the assay.

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents ...

Assessment of Mouse Judgment Bias through an Olfactory Digging Task

Judgment biases (JB) are differences in the way that individuals in positive and negative affective/emotional states interpret ambiguous information. This phenomenon has long been observed in humans, with individuals in positive states responding to ambiguity 'optimistically' and those in negative states instead showing 'pessimism'. Researchers aiming to assess animal affect have taken advantage of these differential responses, developing tasks to assess judgment bias as an indicator of affective state. These tasks are becoming increasingly popular across diverse species and fields of research. However, for laboratory mice, the most widely used vertebrates in research and a species heavily relied upon to model affective disorders, only one JB task has been successfully validated as sensitive to changes in affective state. Here, we provide a detailed description of this novel murine JB task, and evidence of its sensitivity to mouse affect. Though refinements are still necessary, assessment of mouse JB opens the door for answering both practical questions regarding mouse welfare, and fundamental questions about the impact of affective state in translational research.

This article provides a detailed description of a novel mouse judgment bias protocol. Evidence of this olfactory digging task's sensitivity to affective state is also demonstrated and its utility across diverse research fields is discussed.

Judgment biases (JB) are differences in the way that individuals in positive and negative affective/emotional states interpret ambiguous information. This ...

Modeling Early-Life Adversity in Mice Using Limited Bedding and Nesting

Limited Bedding and Nesting as a Model for Early-Life Adversity in Mice

Early-life adversity (ELA), such as abuse, neglect, lack of resources, and an unpredictable home environment, is a known risk factor for developing neuropsychiatric disorders such as depression. Animal models for ELA have been used to study the impact of chronic stress on brain development, and typically rely on manipulating the quality and/or quantity of maternal care, as this is the major source of early-life experiences in mammals, including humans. Here, a detailed protocol for employing the Limited Bedding and Nesting (LBN) model in mice is provided. This model mimics a low-resource environment, which provokes fragmented and unpredictable patterns of maternal care during a critical developmental window (postnatal days 2-9) by limiting the amount of nesting materials given to the dam to build a nest for her pups and separating the mice from the bedding via a mesh platform in the cage. Representative data are provided to illustrate the changes in maternal behavior, as well as the diminished pup weights and long-term changes in basal corticosterone levels, that result from the LBN model. As adults, offspring reared&#160;in the LBN environment have been shown to exhibit an aberrant stress response, cognitive deficits, and anhedonia-like behavior. Therefore, this model is an important tool to define how the maturation of stress-sensitive brain circuits is altered by ELA and results in long-term behavioral changes that confer vulnerability to mental disorders.

Author Spotlight: Exploring Microglial Interactions with Stress-Response Circuitry Using the Limited Bedding and Nesting Model

This protocol describes an animal model for studying how early-life adversity, provoked by an impoverished environment and unpredictable maternal care during the early postnatal period, affects brain development and the future risk of mental disorders.

Early-life adversity (ELA), such as abuse, neglect, lack of resources, and an unpredictable home environment, is a known risk factor for developing ...

Determination of Reproductive Competence by Confirming Pubertal Onset and Performing a Fertility Assay in Mice and Rats

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also termed the hypothalamic-pituitary-gonadal axis. The reproductive axis is a key integrator of environmental and internal input adapting fertility to favorable conditions for reproduction. Prior to embarking upon a fertility study in mice and rats, sexual maturity is evaluated to exclude the possibility that the observed reproductive phenotypes are caused by delayed or absent pubertal onset. This protocol describes a non-invasive approach to assess pubertal onset in males through the determination of preputial separation, and in females through vaginal opening and first estrus. After the confirmation of the completion of puberty and the achievement of sexual maturity, a fertility study can be initiated. The procedure describes the optimal breeding conditions for mice and rats, how to set up a fertility study, and what parameters to evaluate and determine if the treatment or gene deletion has an impact on fertility.

Many treatments and genetic mutations impact the timing of sexual maturity and fertility. This protocol describes a non-invasive method to evaluate pubertal onset in mice and rats prior to setting up a fertility study in sexually mature animals.

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also ...

Basic Care Procedures

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
Mice and rats account for over 90% of the animals used for biomedical research. The proper care of these research animals is critical to the outcome of experiments. There are general procedures that apply to the majority of these mice and rats, but some of the animals, such as the immunocompromised ones, require additional steps to be taken to sustain them for experimentation. 
Commonly used immunocompromised mice include those that have naturally occurred in inbred mice and those that have been created through genetic engineering. The first immunocompromised mice used in research were "nude" mice. The BALB/c Nude (nu) mouse was discovered in 1966, within a BALB/c colony that was producing mice lacking both hair and a thymus. These athymic mice have an inhibited immune system that is devoid of T cells. The value of this animal was soon discovered for the use in studies of microbial infections, immune deficiencies, and autoimmunity. Although not as commonly used as the nude mouse, there is also a nude rat. The nude rat is T cell deficient and shows depleted cell populations in thymus-dependent areas of peripheral lymphoid organs. Another naturally occurring immune deficient mouse is the severe combined immunodeficiency mouse (SCID). These mice have a defect that impairs the production of functional B and T cells. With the inability to mount an adequate immune response, these mice can serve as host recipients for transplants of human cells. Several genetically-modified mouse strains with immune deficiencies are commercially available.1 The severity of the deficiency varies with the genetic modification; however, all of the immunocompromised mice and rats are handled similarly.
This manuscript will describe the care of both immunocompetent and immunocompromised animals. The housing, diet, environmental requirements, and handling will be discussed for both groups of animals.

Basic Care Procedures for Laboratory Rodents

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
Mice and rats account for over 90% of the animals ...