Results

Our original research questions set out to analyze the individual differences in marking behaviors between certain rats, certain times of day, and certain environmental conditions. While the first two questions were simply exploratory and not based in particular theories, previous research of our last question led us to develop a few hypotheses. We hypothesized that rats would demonstrate more marking behavior in conditions with male urine, female urine, burrows, other rats, and the scent of other rats than in a control condition. Many of our original hypotheses were supported while many were not.

Differences in marking behaviors between rats

To analyze the differences in specific marking behaviors between rats, a one-way repeated measures ANOVA was conducted. Results indicate that there is a significant difference between marking preferences of the four rats. Post hoc Tukey tests indicate that there is a difference between the flank marking of Bianca and the flank marking of Suzi, with Suzi marking significantly more than Bianca across all conditions. Rosie also urine marked more than Lemi across all conditions. No differences were found in vaginal marking between rats. Total marking tallies for each rat were not significantly different from each. To answer our original research question, some rats seem to favor certain marking styles but the general quantity and pattern of marking is relatively consistent in all four rats.

Differences in marking behaviors across morning and afternoon trials

As we designed our study, we hoped to control for any changes in a rat’s estrous cycle that might affect their marking behavior. We did this by measuring marking behavior across different times and days. While gathering our data, my partner and I were curious to know whether rats mark their territories differently during the different times of the day. Results from an independent samples t-test indicate that there were no significant differences between total marking behaviors or specific marking behaviors in the morning and evening conditions. Our data suggest that time of day has no significant affect on the marking behaviors of our rats.

Differences in marking behaviors across conditions

The difference in marking behaviors across conditions is the main focus of the present study. Our research indicates that there is a significant difference between the total frequency of marking in the male urine, female urine, and indirect presence condition when compared with the control group, with all rats demonstrating an increased rate of marking across these conditions in comparison with the control condition. Urine marking behavior also differed between the burrow and direct presence conditions and the female urine and direct presence conditions. Vaginal marking behavior differed between the control and male urine conditions, the control and indirect rat conditions, the male urine and control conditions, the male urine and direct rat conditions, the female urine and direct rat conditions and the indirect and direct rat conditions.  In contrast, flank marking differed only between the male urine conditions and the direct rat condition. A table of marking behaviors will be created do demonstrate these differences with better clarity. Generally speaking, the data indicates that male urine, female urine, direct presence, and indirect presence of rat has an affect on the marking behaviors of our subjects. Our results also demonstrate that vaginal marking was most affected by the changes in environmental stimuli.

Discussion

Our initial hypotheses were partially supported by the data and results of this study. The presence of certain environmental stimuli do significantly affect the incidence of marking behaviors in a given location. As expected, the presence of another rat’s urine marking affects the urine and vaginal marking of our test rats. This supports Petrullis’ (1997) original theory that rats have developed an instinctual marking response to other rat urine through evolution.  This also supports the theory that rats respond to the information contained in a scent mark by broadcasting this same information through their own scent mark (Brown & McDonald, 1985). This marking response serves as a social communicative function that transmits information about the animal’s specific species, colony, family, age, sex, reproductive state, and social status (Brown, 1977) and establish social dominance, delineate residential territories, or express sexual receptiveness (Ewer, 1968). When faced with the presence of male or female rat urine, our rats engaged in a scent broadcasting behavior that has been evolutionarily developed over hundreds of thousands of years.

Previous research also provides explanations for the absence of a relationship between marking behavior and the direct rat and burrow conditions. While we expected to see an increase in marking behaviors in these conditions, the absence of a significant marking increase could be due to the fact that the rats chose to engage in locomotive activities instead of exhibiting marking behaviors (Peden & Timberlake, 1990) . In the direct rat condition, the experimental rats exchange information through interactions with the other rat instead of through depositing their scent in the environment. Our surprising results in regards to the burrow condition could be a result of the fact that the observation chamber was a novel and temporary environment, and therefore the rats did not chose to mark the area as a possible reproduction, sleeping, or feeding territory. Future studies should examine how rats appraise an environment for its potential as a residential territory.

Variation in the vaginal markings of the rats can be best explained by the changing estrous cycles of our subjects. While we would like to conclude that all variance in vaginal marking is a result of our experimental manipulations, previous research suggests that vaginal marking changes dramatically in conjunction with a rats estrous cycle (Johnston, 1977, Takahashi & Lisk, 1983). In order to truly demonstrate a causal relationship between environmental stimuli and the occurrence of vaginal marking, researchers would have to control for the effects of changes in a rat’s estrous cycle.

Future research should delve further into the effects of environmental stimuli on an animal’s inclination to broadcast their personal information through vaginal, urine, and flank marking. Researchers have yet to determine the specific factors that contribute to a rat’s assessment of its surroundings. Furthermore, how can our understanding of a rat’s marking behavior translate to a human’s territorial behaviors. Do we have similar evolutionary instincts that motivate us to engage in “marking” behaviors? How and why do human’s choose to delineate residential territories for themselves? Further research is required to fully understand the effects of environmental stimuli on marking behaviors and how these findings influence the human population.

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Burrow Condition

For the burrow condition half a tissue box was placed in the left hand corner of the tank. Two 10 minute sessions were run for each rat (one in the morning and one in the evening) for a total of 8 trials. During each trial researchers observed and noted each type of marking behavior and the number of times each rat exhibited the specified behavior. The graph below illustrates the average number of each type of marking behavior per rat across the two sessions.

marking-averages-for-burrow-condition

Male Urine Condition

For the Male Urine Condition, each rat was exposed to two drops of Male Sprague-Dawley rat urine on a raised plate within the tank. Two 10 minute trials (one in the morning and one in the evening) were conducted per rat for a total of 8 trials. During each trial, researchers observed and noted each type of marking behavior and the number of times each rat exhibited the specified behavior. The graph below illustrates the average number of markings per rat across the two sessions.

marking-averages-for-male-urine-condition

Female Urine Condition

For the female urine condition, each rat was exposed to two drops of female Sprague-Dawley rat urine on a raised plate. The plate was placed in the back left hand corner of the tank. Two 10 minute trials were conducted for each rat (one in the morning and one in the evening) for a total of 8 trials. During each trial researchers observed and noted each type of marking behavior and the number of times each rat exhibited the specified behavior. The graph below illustrates the average number of each type of marking behavior per rat across the two sessions.

marking-averages-for-female-urine-condition

Here is a video showing some of the behaviors we observed throughout our conditions. Specifically this video shows behavior observed during the female urine condition.

Our experiment has progressed substantially this week with the start of the burrow condition. Our team has observed the four rats during two sessions each, one session in the morning and one in the evening. Initial difficulties occurred as we tried to use the same burrow for all four rats. During the first session with the first rat, Bianca marked the burrow itself therefore compromising the experimental control of the subsequent sessions. My partner and I decided to make eight identical burrows for each rat and session. All burrows follow the design outlined in the method section and are constructed from cardboard tissue boxes.

Each rat continued to favor the type of marking behavior they demonstrated in the control sessions. Preliminary t-tests of the results indicate significant differences between marking during control conditions and marking during burrow conditions; the presence of the burrow seems to cause higher instances of each type marking behavior. Means and p-values will be reported after further analysis.

Our next step will most likely be to begin the male or female rat urine condition. We are continuing to consider how to introduce the urine scent and how much to use in each session.  Morning and evening trials will begin during the upcoming weekend.

Here is an example of Flank marking behavior. In the video you can see that Suzi rubs her side up against the walls of the tank. Though it’s brief, it gives a good idea of the behavior we are looking for.

*this video was taken during one of the baseline trials

We ran 2 baseline trials for each of the four rats for a total of 8 trials. Each trial consisted of a 10 minute interval in which all specified marking behaviors were noted. Here is a graph of the averages for each type of marking across the two sessions per rat.

Baseline Marking Averages

This week, my partner and I have started to observe marking behaviors in our four rats: Lemi, Bianca, Rosie, and Suzi. After conducting a pilot test of our method, we decided to include the observation of vaginal marking behaviors in our study so as to increase the possible number of data points for the experiment and to gain a more complete view of marking behavior. Operationalized definitions of each marking variety can be found in our revised method section.

Having improved our method, Jessica and I began to record marking behaviors in the control condition. I observed all four rats in morning sessions at 8:30 a.m. while Jessica observed the rats during evening sessions later in the day. Each rat exhibited marking behaviors on numerous occassions during their 10 minute trial period in the observation tank. Upon preliminary analysis, there seems to be significant variation between the marking behaviors of each rat. For example, Lemi regularly exhibits flank marking while Bianca regularly exhibits vaginal marking and has yet to demonstrate flank marking behavior. Rosie demonstrates a different preference as she frecuently urine marks in the tank during the trials.  This initial observation may lead us to examine individual differences between rats when running statistical analyses at the end of the semester.

Over the next week and a half, we hope to begin observing the rats in the different experimental conditions. To prepare for these sessions, we will order the necesary quantity of male and female rat urine, construct a wire-mesh partition, and design a cardboard burrow. Video updates of our progress will soon be available.

When I was clicker training my rat, I noticed that occasionally she would rub her sides against the walls of the tank. Peden (1990) first defined this behavior as flank marking, noting that rats often press their sides against vertical structures in an attempt to delineate feeding, sleeping, or reproductive territories (Ewer, 1968). Recent research has focused on identifying different variations of female scent marking and situational determinants of increased vaginal and urine marking (Pettrulis, 1997). Operational definitions of vaginal and urine marking have been developed (Dieterlein, 1959; Nelson, 2005) and will be described further in the method section. I decided to focus this research project on studying the situational factors that affect marking behaviors, including flank, urine, and vaginal marking. My research question asks whether the presence of rat urine, rat scent, or a female rat will affect the vaginal, urine, and flank marking behavior of my Sprague Dawley rats.

Method

Animals. Four female Sprague-Dawley rats were used over the course of this study. Each rat had no prior experience with marking experimentation. Two of the four rats used were handled daily for 4 weeks prior to the study and had been trained to be reinforced by a clicker. All rats were housed individually in wire bottom cages and weighed between 205 and 246 grams. The four rats were each fed 20 grams daily and had unlimited access to water. They all stayed within 15 grams of their original weight over the course of the experiment.

Apparatus. A 43.5 x 32.5 x 31.7 cm Plexiglas tank was used to observe the 6 trials. Circular Plexiglas pedestals held the rat scent during all urine conditions. Each pedestal measured 2.5 cm in diameter and 2.5 cm in height. A glass dropper was used to place 2 drops of rat urine on the pedestal. 11 x 11 x 14 cm tissue boxes served as burrows in the last test condition. Each burrow had a 6 x 4 cm doorway in one side of the box. A 31.7 x 32.5cm wire mesh partition was constructed to separate two rats while in the Plexiglas tank for the indirect presence condition.

Procedure. Animals were introduced to the tank for 2 ten-minute trials with no scent or burrow during the control condition. The tank was washed with water and odor remover before and after every trial. Researchers counted each occurrence of flank marking, urine marking, or vaginal marking behavior. Flank marking was defined as rubbing their sebaceous glands of the flank region against a vertical surface. Urine marking entailed lowering their hindquarters and depositing urine on horizontal surfaces. Vaginal marking was exemplified when rats dragged their genital area on the floor of the observation tank. Any instance of these behaviors within a three second period was recorded as one mark. Marking behaviors that occurred more than three seconds apart were tallied as two separate marking instances. Eight control trials with 4 different rats were conducted to establish a baseline reading of marking.

The rats were then observed in each of 6 different test conditions: presence of male urine, presence of female urine, indirect presence of another female rat through mesh, direct presence of another female rat, and presence of a burrow. In both urine conditions, 2 drops of urine were presented on Plexiglass pedestals in the back left corner of the observation tank. The use of a glass dropper ensured that each drop was sufficiently uniform in size. Marking behaviors were observed and tallied with the same method as the control group.

The indirect presence condition was conducted with the use of a wire-mesh partition. Test rats were placed in the observation tank on one side of the wire partition while another female rat explored the other side of the observation tank. Marking behaviors of the test rats were recorded.

In the direct presence condition, one test rat was placed in the observation chamber with one non-test rat. A black dot was placed at the base of the non-test rat’s tail to ensure that the two rats were not confused. Only marking behavior of the test rat was recorded.

Burrow trials used the tissue box structures described above. Each burrow was placed in the back right corner of the observation chamber. Burrows were not secured to the bottom of the tank and marking behavior was recorded in the same manner as the control group.

References

Albers, H. E., Pollack, J., Simmons, W., &  Ferris, C. (1986). A V 1 -like Receptor Mediates Vasopressin-Induced Flank Marking Behavior in Hamster Hypothalamus. Journal of Neuroscience, 6, 2085-2089.

Axelson, J. F. (1987). Scent marking and the maintenance of dominant/subordinate status in male golden hamsters. Physiology and Behavior, 40, 661-644.

Nelson, R. J. (2005). Biology of agression. Oxford: Oxford University Press.

Peden, B. F., & Timberlake, W. (1990). Environmental Influences on Flank Marking and Urine Marking by Female and Male Rats. Journal of Comparative Psychology , 104, 122-130.

Petrulis, A., & Johnston, R. E. (1997). Causes of Scent Marking in Female Golden Hamsters: Specific Signals or Classes of Information? Journal of Comparative Psychology , 111, 25-36.