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 border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Enviro-Dri</td>
			<td colspan="2" style="width:189px;">Shepherd Specialty Papers</td>
 <td></td>
			<td style="width:167px;">Crinkled paper nesting material </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nestlet</td>
			<td>Ancare</td>
			<td style="width:119px;"> </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 ...

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

Research

JoVE Journal

Behavior

26.5K ビュー.  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.

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

The Crossmodal Congruency Task as a Means to Obtain an Objective Behavioral Measure in the Rubber Hand Illusion Paradigm

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden hand is touched. If the viewed and felt touches are given at the same time then this is sufficient to induce the compelling experience that the rubber hand is one's own hand. The RHI can be used to investigate exactly how the brain constructs distinct body representations for one's own body. Such representations are crucial for successful interactions with the external world. To obtain a subjective measure of the RHI, researchers typically ask participants to rate statements such as "I felt as if the rubber hand were my hand". Here we demonstrate how the crossmodal congruency task can be used to obtain an objective behavioral measure within this paradigm.
The variant of the crossmodal congruency task we employ involves the presentation of tactile targets and visual distractors. Targets and distractors are spatially congruent (i.e. same finger) on some trials and incongruent (i.e. different finger) on others. The difference in performance between incongruent and congruent trials - the crossmodal congruency effect (CCE) - indexes multisensory interactions. Importantly, the CCE is modulated both by viewing a hand as well as the synchrony of viewed and felt touch which are both crucial factors for the RHI.
The use of the crossmodal congruency task within the RHI paradigm has several advantages. It is a simple behavioral measure which can be repeated many times and which can be obtained during the illusion while participants view the artificial hand. Furthermore, this measure is not susceptible to observer and experimenter biases. The combination of the RHI paradigm with the crossmodal congruency task allows in particular for the investigation of multisensory processes which are critical for modulations of body representations as in the RHI.

We demonstrate how an objective measure can be employed in the widely employed rubber hand illusion paradigm. This measure is obtained by modifying the well-established crossmodal congruency task. This task allows the investigation of multisensory processes which are critical for modulations of body representations as in the rubber hand illusion.

ラバーハンドイリュージョンパラダイムにおける客観行動測定値を得るための手段としてクロスモーダル一致タスク

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden ...

行動学

Marble Burying and Nestlet Shredding as Tests of Repetitive, Compulsive-like Behaviors in Mice

Obsessive-compulsive disorder (OCD) and autism spectrum disorders (ASD) are serious and debilitating psychiatric conditions and each constitutes a significant public health concern, particularly in children. Both of these conditions are highlighted by the repeated expression of meaningless behaviors. Individuals with OCD often show checking, frequent hand washing, and counting. Children with ASDs also engage in repetitive tapping, arm or hand flapping, and rocking. These behaviors can vary widely in intensity and frequency of expression. More intense forms of repetitive behaviors can even result in injury (e.g.&#160;excessive grooming, hand washing, and self-stimulation). These behaviors are therefore very disruptive and make normal social discourse difficult. Treatment options for repetitive behaviors in OCD and ASDs are somewhat limited and there is great interest in developing more effective therapies for each condition. Numerous animal models for evaluating compulsive-like behaviors have been developed over the past three decades. Perhaps the animal models with the greatest validity and ease of use are the marble burying test and the nestlet shredding test. Both tests take advantage of the fact that the target behaviors occur spontaneously in mice. In the marble burying test, 20 marbles are arrayed on the surface of clean bedding. The number of marbles buried in a 30 min session is scored by investigators blind to the treatment or status of the subjects. In the nestlet shredding test, a nestlet comprised of pulped cotton fiber is preweighed and placed on top of cage bedding and the amount of the nestlet remaining intact after a 30 min test session is determined. Presently, we describe protocols for and show movie documentation of marble burying and nestlet shredding. Both tests are easily and accurately scored and each is sensitive to small changes in the expression of compulsive-like behaviors that result from genetic manipulations, disease, or head injury.

Disease, head injury, genetic modifications, and treatment of mice with drugs can have profound effects on behavior. Utilizing well-characterized and validated approaches such as marble burying and nestlet shredding, compulsive-like behaviors can be documented accurately in mice as models of human obsessive-compulsive disorder and autism spectrum disorder.

マウスにおける反復、強迫様行動の試験などのガラス玉覆い隠しとNestletシュレッダー

Obsessive-compulsive disorder (OCD) and autism spectrum disorders (ASD) are serious and debilitating psychiatric conditions and each constitutes a ...

Quantifying Learning in Young Infants: Tracking Leg Actions During a Discovery-learning Task

Task-specific actions emerge from spontaneous movement during infancy. It has been proposed that task-specific actions emerge through a discovery-learning process. Here a method is described in which 3-4 month old infants learn a task by discovery and their leg movements are captured to quantify the learning process. This discovery-learning task uses an infant activated mobile that rotates and plays music based on specified leg action of infants. Supine infants activate the mobile by moving their feet vertically across a virtual threshold. This paradigm is unique in that as infants independently discover that their leg actions activate the mobile, the infants&#8217; leg movements are tracked using a motion capture system allowing for the quantification of the learning process. Specifically, learning is quantified in terms of the duration of mobile activation, the position variance of the end effectors (feet) that activate the mobile, changes in hip-knee coordination patterns, and changes in hip and knee muscle torque. This information describes infant exploration and exploitation at the interplay of person and environmental constraints that support task-specific action. Subsequent research using this method can investigate how specific impairments of different populations of infants at risk for movement disorders influence the discovery-learning process for task-specific action.

A method is described in which 3-4 month old infants learn a task by discovery and their leg movements are captured to quantify the learning process.

乳児で学習を定量化：追跡脚アクションを発見学習タスクの実行中に

Task-specific actions emerge from spontaneous movement during infancy. It has been proposed that task-specific actions emerge through a discovery-learning ...

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.

豊かな環境の住宅マウス用のプロトコル

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

Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous research determined a profound impact of gentle handling protocols on the well-being of laboratory mice. By introducing clicker training to the keeping of mice, not only do we promote the amicable treatment of mice, but we also enable them to experience cognitive enrichment. Clicker training is a form of positive reinforcement training using a conditioned secondary reinforcer, the "click" sound of a clicker, which serves as a time bridge between the strengthened behavior and an upcoming reward. The effective implementation of the clicker training protocol with a cohort of 12 BALB/c inbred mice of each sex proved to be uncomplicated. The mice learned rather quickly when challenged with tasks of the clicker training protocol, and almost all trained mice overcame the challenges they were given (100% of female mice and 83% of male mice). This study has identified that clicker training for mice strongly correlates with reduced fear in the mice during human-mice interactions, as shown by reduced anxiety-related behaviors (e.g., defecation, vocalization, and urination) and fewer depression-like behaviors (e.g., floating). By developing a reliable protocol that can be easily integrated into the daily routine of the keeping of laboratory mice, the lifetime experience of welfare in the mice can be improved substantially.

The development of new refinement strategies for laboratory mice is a challenging task that contributes towards fulfilling the 3R principle. This protocol introduces clicker training as a cognitive enrichment program for laboratory mice.

実験用マウスのための認知エンリッチメントとしてクリッカートレーニングの紹介

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous ...

Methodology for Establishing a Community-Wide Life Laboratory for Capturing Unobtrusive and Continuous Remote Activity and Health Data

An end-to-end suite of technologies has been established for the unobtrusive and continuous monitoring of health and activity changes occurring in the daily life of older adults over extended periods of time. The technology is aggregated into a system that incorporates the principles of being minimally obtrusive, while generating secure, privacy protected, continuous objective data in real-world (home-based) settings for months to years. The system includes passive infrared presence sensors placed throughout the home, door contact sensors installed on exterior doors, connected physiological monitoring devices (such as scales), medication boxes, and wearable actigraphs. Driving sensors are also installed in participants' cars and computer (PC, tablet or smartphone) use is tracked. Data is annotated via frequent online self-report options that provide vital information with regard to the data that is difficult to infer via sensors such as internal states (e.g., pain, mood, loneliness), as well as data referent to activity pattern interpretation (e.g., visitors, rearranged furniture). Algorithms have been developed using the data obtained to identify functional domains key to health or disease activity monitoring, including mobility (e.g., room transitions, steps, gait speed), physiologic function (e.g., weight, body mass index, pulse), sleep behaviors (e.g., sleep time, trips to the bathroom at night), medication adherence (e.g., missed doses), social engagement (e.g., time spent out of home, time couples spend together), and cognitive function (e.g., time on computer, mouse movements, characteristics of online form completion, driving ability). Change detection of these functions provides a sensitive marker for the application in health surveillance of acute illnesses (e.g., viral epidemic) to the early detection of prodromal dementia syndromes. The system is particularly suitable for monitoring the efficacy of clinical interventions in natural history studies of geriatric syndromes and in clinical trials.

Unobtrusive sensors and pervasive computing technology incorporated into the daily home life of older adults enables meaningful health and activity changes to be recorded continuously for months to years, providing ecologically valid, high frequency, multi-domain data for research or clinical use.

控えめな、継続的なリモート アクティビティおよび健康データをキャプチャするため地域ぐるみライフ研究所を設定する方法について

An end-to-end suite of technologies has been established for the unobtrusive and continuous monitoring of health and activity changes occurring in the ...

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.

全身麻酔を使用してプロシージャのマウスにおける体系的な評価

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

Measurement of Spatial Stability in Precision Grip

The purpose of the protocol is to indirectly evaluate the direction of the finger force during manipulation of a handheld object based on the biomechanical relationships in which deviated force direction causes center of pressure (COP) replacement. To evaluate this, a thin, flexible, and high spatial resolution pressure sensor sheet is used. The system allows measurement of the COP trajectory in addition to the force amplitude and its temporal regulation. A series of experiments found that increased trajectory length reflected a sensorimotor deficit in stroke patients, and that decreased COP trajectory reflects a compensatory strategy to avoid an object slipping from the hand grip in the elderly. Moreover, the COP trajectory could also be decreased by dual task interference. This article describes the experimental procedure and discusses how finger COP contributes to an understanding of the physiology and pathophysiology of grasping.

The goal of this protocol is to measure the center of pressure (COP) replacement using a high spatial resolution sensor sheet to reflect the spatial stability in a precision grip. The use of this protocol could contribute to greater understanding of the physiology and pathophysiology of grasping.

精密グリップにおける空間安定性の測定

The purpose of the protocol is to indirectly evaluate the direction of the finger force during manipulation of a handheld object based on the ...

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.

マウスの行動赤字の初期指標としての巣作り行動

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 Sexual Behavior of Male Mice

Sexual behavior is highly species-specific. Although rodents have slightly different sexual behaviors, mice and rats have a similar sexual behavioral pattern. The purpose of this article is to describe the hormone-induced estrus ovariectomized female model and the experimental procedure for the assessment of sexual behavior of male mice. The most important sexual behavioral elements are demonstrated in the video and illustrations. The critical steps, advantages, and limitations of the sexual behavior test are explained as well. Finally, the behavior parameters are presented, and mounting, intromission, and ejaculation processes in mating are distinguished. Behavioral parameters are assessed in terms of the occurred duration and counts during the test period.

This article describes how to perform sexual behavior tests in male mice.

雄マウスの性行動評価

Sexual behavior is highly species-specific. Although rodents have slightly different sexual behaviors, mice and rats have a similar sexual behavioral ...