Name: a laboratory method to measure contagious yawning in rats
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A. M. was partially funded by the Vicerrector&#237;a de Docencia, Benem&#233;rita Universidad Aut&#243;noma de Puebla. We are especially indebted to the staff of the animal facility &#8220;Claude Bernard&#8221; for the use of the rats for the shoot.&#160;We thank anonymous referees for their comments on early versions of this manuscript. The presentation is less strident and more balanced because of their thoughtful comments

The experimental protocols and animal husbandry were conducted in accordance with institutional guidelines.
1. Materials
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	<li>Find a complete list of the materials used to implement the method in the Table of Materials. Use Figure 1 and seek expert advice to construct an inverted T-shaped table, observation cages, and cage dividers. Follow the safety indications for the use of sharp tools and potentially dangerous materials.</li>
	<li>Make ...

<ol>
	<li>C&#233;zilly, F. A., Danchin, E., Giraldeau, L. A., C&#233;zilly, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+of+Behavioural+Ecology.">History of Behavioural Ecology.</a> Behavioural Ecology. , 3-27 (2008).</li><li>Medina, L. M., Garcia, C. M., Urbina, A. F., Manjarrez, J., Moyaho, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Female+vibration+discourages+male+courtship+behaviour+in+the+Amarillo+fish+(Girardinichthys+multiradiatus).">Female vibration discourages male courtship behaviour in the Amarillo fish (Girardinichthys multiradiatus).</a> Behavioural Processes. 100, 163-168 (2013).</li><li>Mekdara, P. J., Schwalbe, M. A., Coughlin, L. L., Tytell, E. 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L., Gallup, A. C., Vogel, A. R., Vicario, S. M., Clark, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+contagious+behaviors+in+budgerigars+(Melopsittacus+undulatus):+an+observational+study+of+yawning+and+stretching.">Evidence for contagious behaviors in budgerigars (Melopsittacus undulatus): an observational study of yawning and stretching.</a> Behavioral Process. 89 (3), 264-270 (2012).</li><li>Moyaho, A., Rivas-Zamudio, X., Ugarte, A., Egu&#237;bar, J. R., Valencia, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smell+facilitates+auditory+contagious+yawning+in+stranger+rats.">Smell facilitates auditory contagious yawning in stranger rats.</a> Animal Cognition. 18 (1), 279-290 (2015).</li><li>Moyaho, A., Flores Urbina, A., Monjaraz Guzm&#225;n, E., Walusinski, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yawning:+a+cue+and+a+signal.">Yawning: a cue and a signal.</a> Heliyon. 3 (1), e00437 (2017).</li><li>Gallup, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+do+we+yawn?+Primitive+versus+derived+features.">Why do we yawn? Primitive versus derived features.</a> Neuroscience&amp;BiobehavioralReviews. 35, 765-769 (2011).</li><li>Gallup, A. C., Clark, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commentary:+Yawning,+acute+stressors,+and+arousal+reduction+in+Nazca+booby+adults+and+nestlings.">Commentary: Yawning, acute stressors, and arousal reduction in Nazca booby adults and nestlings.</a> Frontiers in Psychology. 6, 1654 (2015).</li><li>Fentress, J. C. <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+grooming+in+mice+with+amputated+forelimbs.">Development of grooming in mice with amputated forelimbs.</a> Science. 179 (4074), 704-705 (1973).</li><li>Nicoletto, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+vision+and+the+chemical+senses+in+predatory+behavior+of+the+skink,+Scincella+lateralis.">The roles of vision and the chemical senses in predatory behavior of the skink, Scincella lateralis.</a> Journal of Herpetology. 19 (4), 487-491 (1985).</li><li>Feh&#233;r, O., Wang, H., Saar, S., Mitra, P. P., Tchernichovski, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+establishment+of+wild-type+song+culture+in+the+zebra+finch.">De novo establishment of wild-type song culture in the zebra finch.</a> Nature. 459 (7246), 564-568 (2009).</li><li>Kapit&#225;ny, R., Nielsen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+yawns+really+contagious?+A+critique+and+quantification+of+yawn+contagion.">Are yawns really contagious? A critique and quantification of yawn contagion.</a> Adaptive Human Behavior Physiology. 3 (2), 134-155 (2017).</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=Social+modulation+of+pain+as+evidence+for+empathy+in+mice.">Social modulation of pain as evidence for empathy in mice.</a> Science. 312 (5782), 1967-1970 (2006).</li><li>Gallup, A. C., Swartwood, L., Militello, J., Sackett, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+evidence+of+contagious+yawning+in+budgerigars+(Melopsittacus+undulatus).">Experimental evidence of contagious yawning in budgerigars (Melopsittacus undulatus).</a> Animal Cognition. 18, 1051-1058 (2015).</li><li>Campbell, M. W., de Waal, F. B., Walusinski, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+Problems+in+the+Study+of+Contagious+Yawning.">Methodological Problems in the Study of Contagious Yawning.</a> The mystery of yawning in physiology and disease. , 120-127 (2010).</li><li>R Development Core Team. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> R: A language and environment for statistical computing. , (2009).</li><li>Moyaho, A., Beristain-Castillo, E., Choe, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Design:+Basic+Concepts.">Experimental Design: Basic Concepts.</a> Encyclopedia of Animal Behavior. 3, 471-479 (2019).</li><li>Urb&#225;-Holmgren, R., 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=Genotypic+dependency+of+spontaneous+yawning+frequency+in+the+rat.">Genotypic dependency of spontaneous yawning frequency in the rat.</a> Behavioral Brain Research. 40 (1), 29-35 (1990).</li><li>Smith, M. L., Hostetler, C. M., Heinricher, M. M., Ryabinin, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+transfer+of+pain+in+mice.">Social transfer of pain in mice.</a> Science Advances. 2, e1600855 (2016).</li><li>Joly-Mascheroni, R. M., Senju, A., Shepherd, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dogs+catch+human+yawns.">Dogs catch human yawns.</a> Biology Letters. 4 (5), 446-448 (2008).</li><li>Barthalmus, G. T., Zielinski, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+skin+mucus+induces+oral+dyskinesias+that+promote+escape+from+snakes.">Xenopus skin mucus induces oral dyskinesias that promote escape from snakes.</a> Pharmacology Biochemistry and Behavior. 30 (4), 957-959 (1988).</li></ol>

There are critical steps in the method that should be taken into account to obtain successful results. Familiar rats must share home cages for at least 1.5 months after weaning and before running the experiments. However, unfamiliar rats must live in separate home cages. In both cases, the pairs of rats must come from different litters but be as similar in age as possible. Regarding the observation cages, their holes should match those in the dividers, because this is the only way to guarantee olfactory contact between t...

Traditionally, communicatory behavior has been studied from two perspectives. From one perspective, ethologists observe and record the behavior of animals in natural settings and attempt to recognize its adaptive value1. The particular sense or senses involved have not been the primary interest of these studies. From another perspective, physiologists are more interested in unraveling the mechanisms by which animals communicate1; hence, laboratory studies have provided methods to address the role that sensory modalities play in communication2,3. These two perspectives are indeed complementary, because knowledge of both adaptive value and immediate mechanisms is necessary to gain a comprehensive understanding of communicatory behaviors in the social life of animals.
Yawning behavior is a conspicuous component of the behavioral repertoire in several species of vertebrates4, ranging from fish to primates5. It can be described as a slow opening of the mouth and maintenance of its open position, followed by a more rapid closure of the mouth5. The duration of the whole sequence depends on the species; for example, primates yawn for longer durations than non-primate species6. In many species, with humans being the exception, males tend to yawn more frequently than females7. This feature might underpin the possible communicatory function of yawning, although regular patterns of yawning and its daily frequency may also suggest a physiological function. In rats, spontaneous yawning follows a circadian rhythm, with peaks of high frequency occurring in the morning and afternoon8,9.
One interesting feature of yawning behavior is that it can be a contagious act (when the releasing stimulus of a behavior happens to be another animal behaving in the same way10) in several species of vertebrates11,12,13,14,15,16, including birds17 and rodents18. Furthermore, recent evidence has indicated that contagious yawning may reflect a communicatory role, because the yawning of one rat can affect the physiological state of another when exposed to olfactory cues19. However, whether or not yawning has a communicatory role is still under debate20,21, and analyzing contagious yawning is an essential first step to solve this issue.
On the other hand, contagious yawning has been linked to an animal&#39;s ability to empathize with the perspectives of other animals; hence, closely related individuals are more likely to show contagion4. This hypothesis has been frequently tested in laboratory conditions in which animals are presented with yawn stimuli on video12,13; hence, contagion can only occur through visual cues. Other investigations have assessed yawn contagion in more natural conditions using groups of animals14,15. A major problem of this is that socially interacting animals often respond to cues and exchange signals that are conveyed through combinations of sensory modalities. Disentangling the actual senses involved in a given behavior from their combined effects is not always an easy task. Typically, researchers pharmacologically or surgically hinder an animal&#39;s use of a given sense, then infer the role of that sense in the relevant behavior2,3,18,22. Fortunately, there are other methods in which only physical barriers are used to either allow or impede communication between animals23,24,25, thereby achieving greater biological validity.
The method proposed here has been specifically designed to study contagious yawning in familiar and unfamiliar rats in a social setting. According to the empathetic hypothesis, the former group should be more susceptible to contagious yawning. The method does not require the animals to be surgically or pharmacologically deprived of any senses. Instead, it works by placing the rats in adjacent cages with holes and physically obstructing their communication using either clear or opaque dividers with or without holes. Thus, four test conditions can be examined: (1) olfactory communication (OC, perforated opaque divider), (2) visual communication (VC, nonperforated clear divider), (3) visual and olfactory communication (VOC, perforated clear divider), and (4) neither visual nor olfactory communication (NVOC, nonperforated opaque divider). Therefore, researchers can compare the relative contributions of olfactory, visual, and to some extent, auditory cues in yawn contagion. This approach is not new, as similar methods have been used to isolate the senses involved in certain communicatory behaviors in animals such as lizards23 and mice26. In fact, Gallup and colleagues27 have used a similar method to demonstrate the role of visual cues in contagious yawning in budgerigars. The main features of these methods are simulation of a social context and the minimal stress inflicted on the animals. Furthermore, the use of interacting animals increases the biological validity of the conclusions.
There are several ways to measure contagious yawning25,28. Dr. Stephen E. G. Lea (personal communication, 2015) helped us numerically adapt a method previously employed by primatologists13,14 for an earlier analysis of the data used here18. Presented in this protocol is an enhanced version of this method with a wider range of applications. It consists of weighting the total number of a rat&#39;s yawns, within and outside of a given time window, by the proportion of observation time corresponding to the yawns within and outside the time window.
For example, if it is assumed that rats A and B are observed for 12 min, their yawning is recorded to the nearest minute, and a 3 min time window is set to measure contagious yawning. Next, the following sequences of yawns for each of those rats are considered: rat A (0,0,0,1,0,0,2,0,0,0,2,1) and rat B (0,1,1,0,1,1,0,0,0,0,0,3). It should be noted that each number (0-3) corresponds to the number of yawns scored at each min. For rat A, during minutes 1, 10, and 11 (numbers in bold type), rat B does not yawn within the preceding 3 min (the chosen time window) or within that minute. In those minutes, rat A yawns a total of 2 times. Therefore, the yawn rate of rat A without any yawn stimulus (non-post-yawn yawn rate) is 2/3 (i.e., 0.67 yawns/min). In the remaining 9 min, rat B yawns at least one time in either the same minute or the 3 previous minutes. Rat A yawns a total of four times in those 9 min. Therefore, the yawn rate of rat A in response to a yawn stimulus (post-yawn yawn rate) is 4/9 (i.e., 0.44 yawns/min). The application of the same procedure to rat B yields a non-post-yawn yawn rate of 2/3 (i.e., 0.66) and post-yawn yawn rate of 5/9 (0.55).
On the other hand, if yawning is recorded to the nearest decimal of a minute, yawn contagion will result in an adjusted post-yawn time. For example, if the following yawn times are recorded over a 12 min observation period for rats A and B: rat A (2.3, 5.1, 5.8, 10.4, 10.8, 11.1) and rat B (1.2, 2.4, 4.5, 5.1, 11.2, 11.6, 11.8). For rat A, the time periods over which rat B does not yawn within the past 3 min range from 0 to 1.2 min and from 8.1 to 11.2 min (i.e., 3.1 min), which yields a total of 4.3 min of non-post-yawn time. The number of times that rat A yawns during those times is three (numbers in bold type), so the non-post-yawn yawn rate is 3/4.3 (i.e., 0.69), while the post-yawn yawn rate is 3/7.7 (i.e., 0.38; the denominator from 12-4.3 min). Similarly, for rat B, the time periods over which rat A does not yawn within the past 3 min range from 0 to 2.3 min and from 8.8 to 10.4 min, which yields a total of 3.9 min. The number of times rat B yawns within those periods is one, so the non-post-yawn yawn rate is 1/3.9 (i.e., 0.25). Accordingly, the post-yawn yawn rate is 6/8.1 (i.e., 0.74).
While a near-contemporaneous match in behavior is an ideal criterion to demonstrate the presence of a contagion, aspects such as the constraints on what an individual attends to, time of reaction to a stimulus, distribution of the behavior over time (e.g., yawning may occur in episodes), and time to acclimatize to the experimental setting all give rise to species differences, making it difficult to use a unique time window. This may be the reason why researchers have used time windows that vary from seconds5 to several minutes11, which creates problems when comparing results28. Because of this, it is proposed to repeat the procedure described above for a range of time windows to obtain yawn contagion curves and compare the yawn contagion curves between species.
Equivalent yawn contagion curves can be compared by randomly distributing the number of yawns observed for each rat over the observation period. Thus, the proposed method to measure yawn contagion offers two types of controls: the (1) yawn rate occurring outside of the time window (non-post-yawn time) and (2) artificial yawn contagion curve obtained from the random distribution of the number of yawns. Therefore, this approach to analyze yawn contagion is a step forward from other procedures, such as those comparing the percentage or frequency of yawning within a single time window to that occurring outside this window25, without taking into account the actual times frames. The method is complemented by an R-based program29 to conveniently and objectively calculate the probability of contagious yawning for one or more time windows.
To illustrate the usefulness of this method and advantages of the R-based program, a data set from a previously published study18 is used. The experimental condition consisted of 144 male rats allocated to either a familiar or unfamiliar condition. The rats in each experimental condition were subdivided into four subgroups of nine pairs and exposed to any of the four test situations described above. The yawning behaviors of the rats in each experimental condition and test situation were then recorded over a period of 60 min.

<table border="0" cellpadding="0" cellspacing="0" style="width:860px;" width="860">
	<tbody>
		<tr height="83">
			<td height="83" style="height:83px;width:221px;">Acrylic dividers</td>
			<td style="width:173px;">Handcrafted</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">Two dividers, one clear and one opaque, will have 24 holes each. The other two dividers, one clear and one opaque, will have no holes. See the main text for details of construction.</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;">An R-based program</td>
			<td style="width:173px;">Benem&#233;rita Universidad Aut&#243;noma de Puebla</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">This is the program used to assess yawn contagion in rats. See the main text for information about the way the program is used.</td>
		</tr>
		<tr height="104">
			<td height="104" style="height:104px;">Data sheets</td>
			<td style="width:173px;">The user can elaborate them</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">These forms will be used for the observer to record the frequency of yawning behaviour by viewing the video recordings. Alternatively, a notebook can be use provided you follow the suggestions given in the main text.</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:221px;">Desktop computer</td>
			<td style="width:173px;">Any maker</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">Make sure the computer has a video card capable of conveniently processing the video recordings of yawning behaviour.&#160;</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:221px;">Digital camcorders</td>
			<td style="width:173px;">Any maker</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">They will be used to video record yawning behaviour of each pair of rats; there will be 2 pairs of rats per experimental session.&#160;</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:221px;">Flash drive</td>
			<td style="width:173px;">Any maker</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">Each experimental session will last 60 min, and so you will require sufficient memory to store the video recording.</td>
		</tr>
		<tr height="125">
			<td height="125" style="height:125px;width:221px;">Glass cages</td>
			<td style="width:173px;">Handcrafted</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">Each cage (19 X 19 X 10 cm height) will have 24 holes (0.5 cm diameter) forming three rows in the middle of one of its sides. See the main text for more details about their construction. It is recommended to fabricate one extra cage in case one of them is accidentally broken.&#160;</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:221px;">Markers</td>
			<td style="width:173px;">Sharpie or any other maker</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">Permanent markers to number the rats. See the main text to see one way of using painting symbols on the rat&#39;s tail.&#160;</td>
		</tr>
		<tr height="104">
			<td height="104" style="height:104px;">Pencils</td>
			<td style="width:173px;">Any maker</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">They are used by the observer to record the frequency of yawning. It is important that the observer has previously been trained to recognize yawning behaviour and operate the video player system.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">R software</td>
			<td style="width:173px;">R Development Core Team</td>
			<td style="width:123px;">Not available</td>
			<td>Download R at: http://cran.r-project.org/&#160;&#160;</td>
		</tr>
		<tr height="104">
			<td height="104" style="height:104px;width:221px;">Rail-like wooden bars</td>
			<td style="width:173px;">Handcrafted</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">They will be fixed in the middle of the rectangular wooden sheet&#160; forming a track, where a second wooden sheet is placed. See the main text for additional instructions for construction.</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:221px;">Rectangular table</td>
			<td style="width:173px;">Any maker</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">This is the table (approximately 2 x 1 m) where the inverted T-shaped table will be placed for performing the observation of yawning behaviour.</td>
		</tr>
		<tr height="146">
			<td height="146" style="height:146px;width:221px;">Sprague-Dawley male rats</td>
			<td style="width:173px;">Any local supplier of laboratory animals</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">Nine pairs of male rats per test situation are necessary for each group, familiar and unfamiliar rats, because with this sample size the interindividual variation that might exist in yawning frequency will not severely affect the conclusions drawn from the statistical analysis performed to the data.&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr height="104">
			<td height="104" style="height:104px;width:221px;">Spreadsheet software</td>
			<td style="width:173px;">Microsoft</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">Excel will be the software used to store the yawning recordings initially recorded on the data sheets. Revise the main text for instructions about the recommended way of doing the transcription.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Square filter papers</td>
			<td style="width:173px;">Any maker</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">They are used for covering the cage&#39;s bottom.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">Tripods</td>
			<td style="width:173px;">Any maker</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">They will be used for fixing the camcorders in front of each pair of observation cages.</td>
		</tr>
		<tr height="125">
			<td height="125" style="height:125px;">Wooden Inverted T-shaped table</td>
			<td style="width:173px;">Handcrafted</td>
			<td style="width:123px;">Not available</td>
			<td style="width:343px;">Read the instructions in the main text to see the way of constructing it. If preferred, a different material to wood can be used. Make sure any material is as resistant as possible to the transmission of ultrasounds, which the rats might use for communication.&#160;&#160;</td>
		</tr>
	</tbody>
</table>

a laboratory method to measure contagious yawning in rats

Communication is an essential aspect of animal social life. Animals may influence one another and come together in schools, flocks, and herds. Communication is also the way sexes interact during courtship and how rivals settle disputes without fighting. However, there are some behavioral patterns for which it is difficult to test the existence of a communicatory function, because several types of sensory modalities are likely involved. For example, contagious yawning is a communicatory act in mammals that potentially occurs through sight, hearing, smell, or a combination of these senses depending on whether the animals are familiar to one another. Therefore, to test hypotheses about the possible communicatory role of such behaviors, a suitable method is necessary to identify the participating sensory modalities.
The method proposed here aims to obtain yawn contagion curves for familiar and unfamiliar rats and evaluate the relative participation of visual and olfactory sensory modalities. The method uses inexpensive materials, and with some minor changes, it can also be used with other rodent species such as mice. Overall, the method involves the substitution of clear dividers (with or without holes) with opaque dividers (with or without holes) that either allow or prevent communication between rats placed in adjacent cages with holes in adjoining sides. Accordingly, four conditions can be tested: olfactory communication, visual communication, both visual and olfactory communication, and neither visual nor olfactory communication. As social interaction occurs between the rats, these test conditions simulate what may occur in a natural environment. In this respect, the method proposed here is more effective than traditional methods that rely on video presentations whose biological validity can raise concerns. Nonetheless, it does not discriminate between the potential role of hearing and roles of smell and vision in yawn contagion.

The rats were selected from a previously produced sub-line of Sprague-Dawley rats that were selected for frequent yawning (approximately 22 yawns per hour31). However, the nine pairs of unfamiliar and nine pairs of familiar male rats (between 2.5 and 3 months of age) used per test situation yawned approximately 12 times per hour, on average18. Therefore, the test situations to measure yawn contagion partially inhibited yawning behavior.
...

Watch this Scientific Journal Video about A Laboratory Method to Measure Contagious Yawning in Rats at JoVE.com

A Laboratory Method to Measure Contagious Yawning in Rats

The method described here aims to obtain yawn contagion curves in pairs of familiar or unfamiliar male rats. Cages with holes separated by either clear or opaque partitions with (or without) holes are used to detect whether visual, olfactory, or both types of sensory cues can stimulate yawn contagion.

Communication is an essential aspect of animal social life. Animals may influence one another and come together in schools, flocks, and herds. ...

Our protocol is significant because it provides an easy way to investigate which senses may be involved in contagious yawning in rats. The use of a non-invasive procedure and the availability of the application software, which speeds the calculation for measuring contagious yawning, are the main advantages of this technique. Individuals who have never performed this technique shouldn't face major difficulties in the implementation, provided they methodically follow each step and seek professional advice when constructing the observation case.Visual demonstration of this method aids in understanding the spatial arrangement of the observation cages during the experiment. Helping to avoid errors in designation of the treatments. Demonstrating that procedure will be Evelyn Beristain-Castillo, a postdoc from my lab.To set up a test session, transfer four rats into the observation room in individual cages. While the animals are acclimating, place an inverted T-shaped table on a larger rectangular table under a ceiling lamp that sufficiently lights the room. Place filter paper on the bottom of each observation cage, and place the cages in pairs on each side of the inverted T-shaped table.Position a divider between each pair of cages, and strategically place two digital camcorders, such that each records the yawning behavior of each pair of rats. After confirming that the camcorders are securely fixed to tripods and correctly oriented to the observation cages, connect the camcorders to a desktop computer to simultaneously monitor the behavior of the rats. When the animals have acclimated for 15 minutes, place them in the observation cages according to the previously determined allocation, and set the automatic focus to off on each camcorder.Start the video recording and a stopwatch at the same time, stopping the video recording at the end of the 60 minute observation period. When the observation is over, return the rats to their home cages in the animal facility with access to food and water. Then thoroughly clean the observation cages with a non-toxic detergent before preparing the experimental set up to perform the subsequent test sessions for the day.After the last session, play back each video on a computer screen using any standard playback system for an observer. Have the observer first view each video to observe and record the yawning behavior onto data sheets once at normal speed, followed by additional viewing at slower speeds to make it easier to observe and measure the yawning. To score the yawning, use vertical lines to represent the occurrence of a yawn with the minute written as a superscript.When all of the yawns have been scored, transcribe the temporal sequence of yawns in each rat to a spreadsheet. If the yawning was recorded to the nearest minute, enter the number of yawns at each relevant minute and use zero to fill in the cells when no yawn occurred in a given minute. Then save the worksheet as a text file.If the yawning was recorded to the nearest decimal of a minute, type in the sequence top down. Use NA to fill in the empty spaces of the rat columns in which the number of yawns in the rows is lower than that of the column. With the maximum number of rows of yawns recorded for a rat.Then save the worksheet as a text file. When all of the data have been entered, import the text file into R.Save the program codes with the extension code R and download the specific program code depending on whether the yawns were recorded as integers or fractional numbers. Then run the program for each pair of rats and the desired number of time frames.At the end of the run, run the program again using a random distribution of the number of yawns from each rat. To create yawn contagion curves, use the data previously saved in the spreadsheet to subtract the non-contagion rates from the contagion rates for each pair of rats, the time window, and the test situation, separating the analyses of the observed data and artificial data. In this representative experiment, the yawn contagion between unfamiliar male rats was measured over a range of time windows that varied from one to 10 minutes.These yawn contagion curves indicate that only olfactory communication between rats in cages with a perforated opaque divider exhibited yawn contagion. Which is evident from the time window four and onward, as the confidence interval of the averages does not overlap with the confidence interval of the randomly allocated number of yawns over the 60 minute period. The band indicated, as expected, that a random allocation of the number of yawns in each rat in the observation period, produced yawn rates that oscillated at approximately zero without demonstrating any apparent pattern.In this representative experiment with familiar male rats, none of the four test situations stimulated yawn contagion because their confidence intervals overlapped with the randomly generated confidence interval band. Further, there were no differences among the four test situations even though the overall increase in yawn contagion over the 10 time windows different from a zero slope. Remember to keep track of the familiar and unfamiliar identities of the rats for a correct interpretation of the results, and to clean the observation cages between experiments.A pharmacological procedure may be used to block auditory communication, and to completely separate the hearing effects on contagious yawning from the effects from the other senses. This technique has paved the way for comparing contagious yawning across the species both in terms of the senses involved, and the strength of the response by comparing the contagious yawning curves. Remember to carefully handle the observation cages because they can accidentally break and cause injuries.

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