Observation and Measurement of Behavior of Mus musculus1

Marie Arenbi Carillanes Eunice Marie Jaen Arriane Mae Isla Ivy Madrid Paulo Miguel Kim

A scientific paper submitted in partial fulfillment to the requirements in Animal Behavior Laboratory under Mr. Pablo Ocampo, 1st sem, 2011-2012.

INTRODUCTION In a purely biological perspective, behavior is defined as all observable muscular and secretory responses to changes in an animals internal or external environment (Grier, 1984). However, such a definition is far too broad. Behavior can be encompassed by physiology, psychology and sociology. But it can also be tackled in the field of zoology. In its simplest form, one can consider behavior as the collection of observable and recognizable actions of animals. Any study of animal behavior always starts at the same line. By far it is the most important yet also one of the most overlooked. A thorough knowledge of the target specimen being studied is a critical necessity for any behavioral studies. It ultimately will lead to a proper citation and formulation of a special behavioral set unique to a species, a list known as an ethogram. An ethogram is a repertoire composed of sequences of behavioral patterns. Each pattern in turn is composed of ethons, the smallest unit in the study used to denote the tiniest action of behavior which makes up the whole (Ibid.). In dissecting the total behavior displayed by an animal, one can derive the ethons and attribute the correlations of each in forming possible conclusions to their existence. Some behavior of animals are innate in nature, a necessity for survival (Breland, 1966). But because behavior is variable, it cannot be expected that the different animals will display the same behavior. Thus, a large amount of time is used just to document the list of behavior of a single animal and a greater amount of patience is employed to ensure that the list of behavior is as close to the animals. However, the number of acts that can be observed in an animals repertoire is dependent in three factors. First, one must consider the number of acts that an animal possesses. Second, the rarity of a certain act within a bout must also be considered, whether or not observers will be able to ascertain such behavior. Lastly, one must consider the duration of observation. The longer one observes the higher probability of seeing the rarer acts. Behavior can be grouped accordingly in a logical, natural categories based on their function. They can also be illustrated according to the measurement of the continuous aspects of an animals appearance (i.e. posture, angle or position on limbs) (Grier, 1984). Statistics provide studies in behavior the necessary tools in order to understand the relationship of the observations made. Descriptive statistics aid in describing the characteristics of a set of numbers. Inferential statistics on the other hand, is used when one infers, with the aid of assumptions and statistical tests like analysis of variance, how groups of data relates to each other and if whether there is an inherent order or variability. Between the two, inferential statistics is more highly used, the former often regarded as existing under the auspices of random

change rather than by any underlying effect of interest.

Often times, behavior is taken is as part of a multiple whole rather than as a single entity. Among the common categories of analyzing multiple behavior data are the comparison of the frequency of behavior among different rest subjects, the analysis of the interaction of behavior between two or more individuals and the analysis of the sequence of behavior through time. Statistical analysis is further subdivided into two types: parametric analysis, where the assumption that a population is normally distributed and variance is assumes homogenous and non parametric, where it is not necessary for a population to be normally distributed or for variance to be homogenous(Changing Minds, 2011). The common house mouse (Mus musculus) is a common test animal in the study of behavior. It exhibits a variety of behavior and is easy to monitor as well as manipulate. It is an efficient subject particularly for observing behavioral patterns and for detecting ethons within series of complex acts. Grooming behavior is among the most observed acts among mice and an ideal study for sequential behavior. The objectives of the experiment is to study the grooming behavior of animals, specifically Mus musculus, Furthermore, the experiment will ascertain the grooming behavior of Mus musculus under stress and to determine the significance of the behavioral pattern and its subunit suingthe following measures: duration, interval, latency and sequence. In the study, the principles of observation, description and quantification of behavior will be performed and evaluated. This study was conducted at A-125, Institute of Biological Sciences, UPLB on August 2011.

METHODOLOGY In ascertaining the grooming behavior of mice, an observation chamber set up was assembled. Three aquarium tanks were setup. Fluorescent light was wrapped in red plastic to mask the light. The sides of the aquarium were covered to delimit the red light within. Three mice (an individual, a male and a female) were produced. The individual test mouse was coated in flour inside a plastic bag and allowed to acclimatize in the observation chamber for 5 minutes. The grooming behavior of the mouse was complied and labeled into specific codes for later use. Observation was made for 15 minutes. The test mouse was redusted and reintroduced to the chamber. The procedure was repeated for another 20 minutes. The grooming acts were noted using the formulated codes and the actual time until the end of grooming was recorder in seconds. All data and observations was tabulated (Table 2). The male and female mice were tested separately. Each test mice was sprayed with copious amount of water and placed in the chamber. Using the previously formulated codes, all grooming acts displayed were noted in a sequence for 30 minutes at a one minute interval. All data and observations were tabulated (Table 3). The mean of the duration, interval and frequency of the first experiments were computed and tabulated. Similarly, the mean frequencies of the male and female mice for the second experiment were also computed. All computed measures were pooled. The Kruskall-Wallis Single Factor ANOVA was applied. Results were analyzed and predominant sequences were established from the data.

RESULTS AND DISCUSSIONS The data was subdivided into two main experiments. An initial set up was established to observe grooming subunits as well as to establish a sequence code for later use (Table 1). The first experiment involved the use of an individual mouse placed into a condition to elicit its grooming behavior, the condition here is the use of flour as a stimulus. Two sets were made, the first to ascertain the categories and the subunits of its behavior based from observation. The second set was implemented to establish the sequence of the behavior being displaced under 15 minutes and form a conclusion based on the resulting frequency, interval and duration of the behavior (Table 4., Table 5., Table 6.) The second experiment involved two specimens, a male and female mouse. Similarly to the previous, the experiments involved subjecting the specimens into specific conditions which would elicit their grooming behavior. However, unlike them former, water was used instead to ascertain the resulting behavior. The previously established categories and subunits were also applied in the experiment as the same procedures. However, only the frequencies of the categories were taken. (Table 7., Table 8. ) The results of the first experiment showed that, of the four categories of grooming (Head, Body,Coxal and Leg), the longest duration as well as interval for the two specimens was for body grooming (8.36 and 8.2 seconds respectively) for each bout. On the other hand, the most frequency grooming category exhibited was seen in leg grooming, with a mean total of 26.5. The results of the second experiment, on the other hand, showed that for males, there was higher average frequency of leg grooming in contrast to the other categories as well as those of the female (34.25). The female specimens, however, exhibited a higher average frequency of head grooming against the other categories (27.25). In order to establish the significance of the data set, a non-parametric statistical tool was employed. Instead of using single factor ANOVA, Kruskall-Wallis single factor ANOVA was used. The Kruskall-Wallis one-way analysis of variance uses ranks of the measurements instead of the actual measurements of variabilities of the populations being compared. It is an extremely useful test on deciding whether k independent from different populations. Furthermore, the test assumes that the variable under study has an underlying continuous distribution (Table 9). Using the Kruskall-Wallis single factor ANOVA, the following results were obtained: 1. With H values of 3.17, 3.17 and 4.17 respectively, it was concluded that there was no statistical significant differences among the durations, intervals and frequencies of the four grooming behaviors in the two mice specimens.

2. With H value of 5.41, it was ascertained that there was no significant, statistical differences between the frequencies of the four grooming habits in the four male mice specimens. 3. With a H value of 7.18, it was determined that there was no significant statistical differences between the frequencies of the four grooming habits in the four female mice specimens. From the results of the experiments, it can be established that there is not significant differences in the different grooming habits of the mice. As such, although some grooming categories and subunits were exhibited more within a single bout than others, there is no statistical evidence that there is a constant, established pattern being followed. However, from the results of the experiment, it was found that in terms of frequency, leg grooming and its associated subunits was potentially higher than those of the other categories. But in terms of both duration and interval, body grooming was exhibited longer and within greater intervals. It will also be noted that head grooming closely followed both of the previous, acting as an intermediate in terms of frequency, duration and interval. While it cannot be sufficiently established from the results, it may postulated that the after mentioned three categories of grooming were more exhibited in a given bout. Coxal grooming proved to be the least exhibited and in fact has the lowest values in terms of the three parameters. Anxiety and stress often plays a major part in grooming amongst many mammals, especially among mice. Depending on the level of stress, grooming may be erratic, passive or robust (Kyzar et al., 2011; Animal Behavior Society, 2011). In mice, the more anxiety experienced, the more vigorous grooming is. With the setup of the experiment, stress is reduced by a combination of acclimatization and the red light that prevents the specimens from detecting the observers. However, stress was added in order to elicit grooming responses, namely subjection to flour and water, as seen in the first and second experiment respectively. Thus, it was expected that the mice were to exhibit vigorous grooming behavior (Kyzar et al., 2011). In the flour stimulus experiment, although leg grooming was more frequently exhibited at a bout, body grooming was performed at a longer duration as well as in greater interval. As was previously mentioned, head grooming was considered at an intermediate position between the two. Coxal grooming was not emphasized nor displayed recurrently. In the water stimulus experiment, while the duration and interval were not noted, the frequency of the male and female mouse were ascertained. The male mouse showed an higher

average frequency of leg grooming (34.25), followed by head grooming (27.25) whereas the female showed a higher average frequency of head grooming (27.25), followed by leg grooming (24.5). Coxal grooming was low in terms of frequency, for both male and female mice. Of the four grooming categories, the head and body grooming were more emphasized by mice due to the inherent consequences associated with keeping the two regions free from irritants. The head region involves many of the senses relied upon by mice in order to cope with its environment. Likewise, the body is necessary in order to maintain coordination among mice, an important trait for survival. On another note, the leg region, though only frequent in duration in the experiment, also is an important area which must be maintained by mice, being its essential tool for locomotion. If it is thus analyzed, the head region houses the vital sense organs like the eyes, ears and nose. These sense organs are used by mice in order to survey their environment for any subtle changes that may signal for security as well as for detecting food. Irritants that impede the functions of these senses must be removed to maintain optimum use of these senses. On the other hand, once the brain processes the stimuli, it must act depending on the significance of the message, whether to maintain its position or to escape. Likewise, the body as well as the legs must be groomed to avoid critical mistakes in its behavioral responses. Irritants that block the optimum use of the body and limbs could prove a fatal mistake for many a species. However, in mice, it was observed that the coxal grooming was the least emphasized grooming category and the least observed in a particular bout. It may be postulated that coxal region, a region whose usual function is to maintain gait during locomotion, is least displayed because the tail poses no immediate role in behavioral mechanisms like escape and detection. Thus, even in a passive environment where the levels of threat are low, the mice specimens perform coxal grooming less frequently as compared to grooming the other regions. Lastly, it must be noted that the tail, unlike most of the other body regions, has relatively sparse fur and thus least likely to be affected by irritants like dirt and parasites. There is a distinguishable sequence which maybe observed within the grooming acts. This sequence is best seen in the second experiment where the male and female mice were involved. As can be seen in Table. 2 and 3, there is a complex number of apparent sequences which may be observed. However, segments of the major sequence of grooming behavior can still be noted. It will be noted that majority, if not all, of the grooming acts per one minute were initiated by head grooming. Although no true pattern could properly be established, the head grooming subunits began with licking or scratching different to successive parts of the head.

After head grooming, body or leg grooming followed. However, between the two, leg grooming proceeded first before body grooming, though instances arose where in the latter appeared in the absence of the former. The grooming step usually proceeded from the leg to the sides of the body, gradually progressing ventrally until the anal-genital regions was reaches. Finally, observations yielded that a singular pause was usually performed before proceeding to coxal grooming. In general, a grooming sequence could be established: Head-Leg-Body-Coxal. However, it will be noted that grooming of the coxal region was infrequent and thus, there were only few instances where in the entire sequence were actually observed. Other than that, there were frequent recurrent fragments of this major detectable sequence. Each fragment follows the established sequence closely and with little to no deviation. The after mentioned grooming sequence that was established by the experiment is common to all rodents. All rodents engage in a number of self-grooming activities in order to keep the fur and the skin clean. The form in which these sequences as displayed may differ from one rodent to another but the basic pattern is retained. Other times, the organization varies from very loosely to very stylized though always in an identical fashion. This pattern is an extensive grooming activity known as cephalocaudal grooming. This grooming sequence, begins from the face (head region) and gradually covers up to the flanks (the leg and body region) and finally to tail (coxal region). Grooming, however, may be interrupted from between points in the sequence, most notably the abrupt pause before tail grooming (Rat Behavior, 2008; 2011).

SUMMARY AND COCNLUSION The observation and measurement of behavior of the albino variant house mouse (Mus musculus) has been determined. The compilations of a behavioral catalogue for the specimens as well as the dissection of the individual bouts have yielded four major grooming categories (Head, Leg, Body and Coxal), each of which were further subdivided into subunits. Results of the initial experiment on individual test mice showed that leg grooming , with a mean frequency of 34.25, showed the highest frequency displayed whereas the longest duration and the greatest interval was shown in body grooming (8.36 and 8.2 seconds respectively). Results of the second experiment showed that the highest average frequency of grooming behavior in males was under leg grooming (34.25) whereas the highest average frequanecy for female mice was under head grooming (27.25). Using Kruskall-Wallis Single Factor ANOVA, it was determined that there were no significant statistical differences between the duration, interval and frequency of the four grooming acts of the individual mouse in the flour test. Similarly, there were no significant statistical differences between the frequency of the four grooming behavior of both the male and female mice in the water test. Thus, there is no direct correlation that a grooming act on a certain body part is exhibited more than the others. Lastly, a detectable sequence was established from the resultant data. A pattern of HeadLeg-Body-Coxal grooming was determined to persist in any series of behavior displayed by the test mice, a main sequence which is followed whether whole or in fragments. Furthermore, the cephalocaudal grooming displayed was loosely organized, the intervals often interrupted, thus giving rise to the fragments of the main sequence. The measurement of behavior is mediated by the careful use of observation as well as statistical analysis in order to ascertain significance. In the study of ethograms, the initial step is to determine the smallest identifiable units of behavior by dissecting the observable behavior of an animal. Behavioral patterns can be determined after following the complex sequences formed from these units. In the case of the test mice, the smallest units that make up the entire behavioral pattern are ultimately responsible for the overall makeup of the sequence. By determining parameters such as duration, interval, latency and frequency, it is possible to determine a possible base sequence of behavior.

REFERENCES Animal Behavior Society. Ethogram of Mice. <http://www.animalbehavior.org/ABSEducation/laboratoryexercises-in-animal-behavior/laboratory-exercises-in-animal-behavior-ethograms>.

Accessed August 2011.

Breland, K. and M. 1966. Animal behavior. New York: The Macmillan Company.
Changing Minds. Parametric vs. non-parametric tests. <http://changingminds.org/explanations/research/analysis/parametric_non-parametric.htm>. Accessed August 2011.

Grier, J. W. 1984. Biology of animal behavior. St. Loius, Mo.: Times Mirror/Mosby College Publishing. Kyzar, E., Gaikwad, S., Roth, A., Green, J., Pham, M., Stewart, A., Loang, Y., Kobla, V. and Kallueff, A. 2011. Towards high-throughput phenotyping of complex patterned behaviors in rodents: Focus on mouse self-grooming. New Orleans: Elsevier B.V.
Rat Behavior. 2008. Rat Behavior and Biology. <http://www.ratbehavior.org/norway_rat_ethogram.htm>. Acessed September 2011. ---------------2011.Glossary of rat behavior terms. < http://www.ratbehavior.org/Glossary.htm>. Acessed September 2011.

Siegel, S. 1956. Non-parametric statistics for the behavioral sciences. USA: McGraw-Hill Book Co.

APPENDIX
Table 1. Catalog listing of grooming behavior. Grooming Act Lick arm Lick leg Lick feet Lick ear Lick nose Lick cheek Lick head Lick back Lick stomach Lick testicle Lick base tail Lick middle tail Lick tip of tail Subunits Scratch arm Scratch leg Scratch feet Scratch ear Scratch nose Scratch cheek Scratch head Scratch back Scratch stomach Scratch testicle Scratch base tail Scratch middle tail Scratch tip of tail LLA LLL LLF HLE HLN HLC HLH BLB BLS BLT CLB CLM CLT Codes LSA LSL LSF HSE HSN HSC HSH BSB BSS BST CSB CSM CST

Leg

Head

Body

Coxal

Table 2. The sequence of grooming acts in terms of duration and interval. Succession of Grooming Acts 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Gooming Act (coded) Beginning time (s) End time (s) HLH 5:05 5:05 LLF 5:06 5:06 HLH 5:06 5:06 HLH 5:06 5:06 HSN 5:06 5:07 LSF 5:08 5:08 LSF 5:08 5:08 BLS 5:08 5:09 HLH 5:09 5:09 LLF 5:09 5:09 HLH 5:09 5:09 HLH 5:09 5:10 LLF 5:10 5:10 LLF 5:10 5:10 BLB 5:10 5:10 LSF 5:10 5:10 HLH 5:10 5:10 BLT 5:10 5:10 LLF 5:11 5:11 BLS 5:11 5:11 BLS 5:11 5:12 BLB 5:12 5:12 LLF 5:12 5:12 HLN 5:13 5:13 LLF 5:13 5:13 LSF 5:13 5:13 LLF 5:13 5:14 HLC 5:14 5:14 BLB 5:14 5:15 HSN 5:16 5:16 BLS 5:16 5:16 LLF 5:16 5:16 Duration (s) 15 2 3 2 3 2 2 3 6 5 3 2 3 4 7 3 3 5 10 8 4 16 11 3 7 4 10 3 9 15 14 5

33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59

HLH BLS BLT HLH BLT HLH BLB BSB LLF LSF BLS HSN LLF HSE HSE HSN HSN HSN BLS HLH HLH HSC BLS LLF HLN LLF LLF

5:16 5:16 5:17 5:17 5:17 5:17 5:18 5:18 5:18 5:19 5:19 5:19 5:19 5:20 5:20 5:20 5:20 5:20 5:21 5:21 5:22 5:22 5:22 5:23 5:23 5:24 5:25

5:16 5:17 5:17 5:17 5:17 5:18 5:18 5:18 5:18 5:19 5:19 5:19 5:20 5:20 5:20 5:20 5:20 5:21 5:21 5:22 5:22 5:22 5:23 5:23 5:23 5:25 5:25

2 12 7 4 5 6 13 12 7 5 9 3 9 3 4 2 3 2 8 4 5 2 14 8 5 6 12

Table 3. The sequence of grooming acts of male and female mice. Animal 1 (male) No . 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Grooming Acts (Coded) HSM, HLN, HLH, HSC, HSE, LSF,LLF HSC HSN, LLF HLH, LLF, BLS HLH,HSN, HLC, LLF, BLS HSB, LLF, BLB HSE, LLF, LLH, BLB HSE, HSN, HSC, LLF, BLB,BLT HLH, HSN, BLB, HSN, LLF, BLS HSN, HLH HLH, HSE, BLS, BLB HLH, LSF, BLB, CEB HSN, HLH, BLB HLH, HSE, BLB HLH, HSE, BLB HSN, LLF, BSB, CLB HSC, HSN, HLOH, LLF, BLS, BLB HLH, BLT, BLB HSE, HSN,LLH, BLB, CLB HSE, HLN, HLH, HSE, HSC HSE, HSN, HSC, BLB, BLS BSB, BSS, BLT HLN HSC, HLN, HSE, BLB, BLT BLT HLH, BLT, BLB HLH, LLF, BSS, BLB, BLT, CLB No . 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Animal 2 (Female) Grooming Act (Coded) HSN HSN, HSE, LSF, BSB HSE, HSN, HSC, LSF, LSF, BSB, BLB HSE, HSC, HSE, LLF, LLF, BSB HSF,BLS, BLB,CLB HSF, LSF HSN HSF HSE, HSN, HSC, HLE, LSF,LLF HLN, HLE, HSE, HSE, HSN, LLF, BLG, BLS, BLB HLH, HLN, BLB, BLG, BLS HLE, HLC, HLH, HLN, LLF, BLB, BSB, CLB HSE, HSN, LLH, LLF, BLB, BSB, BLG, BLB HLH, HLN, HSE, HSC, BLS, BLG, BLB HLH, BSB HSN, HSE, HSC, LLF, BLS, CLB HSN, HSE, HSC, LLH, BLS HSE, HSC, BLS, BLG, CLB LLH

Table 4. Summary of pooled data of the average duration of grooming in individual mice. Mouse no. 1 2 mean sd HG 4.29 8.15 6.22 2.729432175 LG 6.05 5.76 5.905 0.205060967 CG 0 6.25 3.125 4.419417382 BG 9.13 7.59 8.36 1.088944443

Table 5. Summary of pooled data of the average intervalof grooming in individual mice. Mouse no. 1 2 mean sd HG 7.5 7.68 7.59 0.127279221 LG 8.77 5.8 7.285 2.10010714 CG 0 6.45 3.225 4.560838739 BG 9.33 7.07 8.2 1.598061325

Table 6. Summary of pooled data of the average frequency of grooming in individual mice. Mouse no. 1 2 mean sd HG 24 26 25 1.414213562 LG 19 34 26.5 10.60660172 CG 0 4 2 2.828427125 BG 16 29 22.5 9.192388155

Table 7. Summary of pooled data of the average frequency of grooming in male mice. Mouse no. 1 2 3 4 mean sd HG 1 57 50 1 27.25 30.44530615 LG 19 83 19 19 35 32 CG 0 13 4 0 4.25 6.130524719 BG 10 31 33 10 21 12.72792206

Table 8. Summary of pooled data of the average frequency of grooming in female mice. Mouse no. 1 2 3 4 mean sd HG 13 40 43 13 27.25 16.5 LG 16 52 14 16 24.5 18.35755975 CG 5 6 4 5 5 0.816496581 BG 10 27 26 0 15.75 13.07351011

Table 9. Summary of results of Kruskall-Wallis computations. Table no. 4 5 6 7 8 k value N value Tabulated value H value 4 4 4 4 4 8 8 8 16 16 6.167 6.167 6.167 7.235 7.235 3.17 3.17 4.167 5.41 7.18 Decision Fail to reject Ho Fail to reject Ho Fail to reject Ho Fail to reject Ho Conclusion No significant differences No significant differences No significant differences No significant differences

Fail to reject Ho No significant differences